Original article
Induction of pro-inflammatory cytokines by human T-cell leukemia virus type-1 Tax protein as determined by multiplexed cytokine protein array analyses of human dendritic cells

https://doi.org/10.1016/j.biopha.2007.02.006Get rights and content

Abstract

Human T-cell leukemia virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is characterized by a hyperstimulated immune response, including elevated levels of inflammatory cytokines/chemokines and oligoclonal expansion of virus-specific CD8+ T cells in the cerebrospinal fluid. Studies have shown that the HTLV-1 transactivator protein Tax is available for immune recognition by antigen presenting cells (APCs), such as dendritic cells (DCs). DCs are relevant to the pathogenesis of HAM/TSP because the presentation of Tax peptides by activated DCs to naïve CD8+ T cells may play an important role in the induction of the Tax-specific immune response that is observed in HAM/TSP. In this study, a human cytokine protein array was used to study the secretion of cytokines by monocyte-derived DCs (MDDCs) exposed to Tax. Of the 16 cytokines analyzed, 6 cytokines were secreted in significantly high amounts (≥2-fold), including Th1 cytokines (IFN-γ, IL-12, and TNF-α) and C-C chemokines (Eotaxin, MCP-1, and MCP-3). Selected cytokines were further examined at two concentrations of Tax and at two time periods. Furthermore, a transient exposure to Tax did not result in any cytokine production when examined at three different time points after exposure, indicating that a prolonged presence of Tax is required for its activity. Finally, inhibition of the NF-κB signaling pathway by specific inhibitors, abrogated Tax-mediated cytokine secretion. Collectively, these findings suggest a role for Tax-induced cytokine secretion from MDDCs, which may be critical for the cellular activation and tissue damage that has been observed in HAM/TSP.

Introduction

Human T-cell leukemia virus type-1 (HTLV-1), an exogenous type C retrovirus, infects 10–20 million people worldwide [1] and is endemic in southern Japan, Caribbean, Central and South America, Middle East, and Africa [2]. While the majority of infected individuals are asymptomatic carriers, about 2–3% develop a progressive lymphoma designated adult T-cell leukemia (ATL) and an additional 0.5–3% develop a debilitating neurological disease known as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) [3]. HAM/TSP is a chronic progressive disease of the central nervous system (CNS) that is characterized by weakness and stiffness in the lower extremities, lower back pain, urinary dysfunction, thoracic myelopathy, and paraplegia. Pathologic observations indicate a symmetrical loss of myelin and axonal dystrophy in the thoracic and lumbar regions of the spinal cord [4].

It is assumed that the CNS tissue damage, which is observed in HAM/TSP, is the result of a hyperstimulated immune response and several theories on how this immune response may contribute to tissue damage have been proposed [5]. One theory suggests that HTLV-1 may infect neurons or CNS resident cells and that these infected cells may, in turn, be subject to HTLV-1-specific immune responses. Other theories focus on the mechanism of CNS damage and assume that antibodies or T-cells, which recognize certain HTLV-1 epitopes, may cross-react with the host cell heterogenous nuclear ribonucleoprotein A1 to promote antigen mimicry [6], [7]. An additional pathogenic mechanism, which is likely to operate in HAM/TSP, involves the process of “bystander” or “collateral” damage caused by inflammatory cytokines released by HTLV-1-infected cells that may have crossed the blood–brain barrier (BBB) [8].

Cytokines play pivotal roles in the regulation of the immune response and the HTLV-1 transactivator protein Tax has been postulated to contribute to the inflammatory response observed in HAM/TSP as a result of the aberrant regulation of host cell cytokines [9]. Elevated levels of pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), interleukin 1 (IL-1), IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF), have been detected in the cerebrospinal fluid of HAM/TSP patients [10], [11], [12]. Supporting the hypothesis of “bystander” damage, it has been suggested that the HTLV-1-induced damage caused to the CNS involves an immune-mediated disease in which Tax can impart some of its pathological effects by functioning as an extracellular cytokine [13], [14], [15], [16], [17]. The presence of cell-free Tax in cerebrospinal fluid (CSF) has been demonstrated during progression to HAM/TSP [18] and may be the result of apoptosis or necrosis of HTLV-1-infected cells or the secretion of Tax from Tax-expressing cells [19]. Extracellular Tax has been shown to induce production and secretion of TNF-α from a human neuronal cell line [20]. Primary human microglial cells have also been shown to respond to extracellular Tax by secreting inflammatory cytokines TNF-α, IL-1β, and IL-6 [21]. Tax-stimulated primary human peripheral blood macrophages have also been shown to produce and secrete TNF-α, IL-1, and IL-6 [21] and soluble Tax treatment has been shown to induce the nuclear accumulation of NF-κB, as well as the expression of the immunoglobulin kappa light chain [13], [15], TNF-β [13], and IL-2 receptor in lymphoid cells [17].

Bystander damage to the CNS tissue in HAM/TSP likely involves the concerted activity of several infiltrating immune cell populations. Although CD4+ T cells, dendritic cells (DCs), and monocytes/macrophages contribute to the pathogenesis of HAM/TSP, the CD8+ T cell compartment plays an important role in neurodegeneration, as reflected by the oligoclonal expansion of Tax-specific cytotoxic CD8+ T cells (CTLs) in the CSF of infected individuals [6]. Since the induction of a CTL response is strictly dependent on antigen presentation and co-stimulation provided by antigen presenting cells (APCs), it is hypothesized that these immune cell components are critical for the genesis of a Tax-specific host immune response. In support of this hypothesis, the most potent APCs, dendritic cells, obtained from HAM/TSP patients, have been shown to be infected with HTLV-1 and to stimulate autologous T-cell proliferation [22], [23], [24]. DCs exposed to extracellular Tax have also been shown to undergo activation and maturation [25], [26] and may thereby facilitate the entry of HTLV-1-infected cells into the brain by compromising the integrity of the BBB through the secretion of inflammatory cytokines and chemokines, contributing to the tissue damage observed in the CNS of HAM/TSP patients. We have previously shown, by utilizing gene expression analyses with a pathway-specific gene array, a range of pro-inflammatory cytokines and chemokines that were differentially expressed following treatment of DCs with extracellular Tax [25]. However, very few protein-based investigations of the pro-inflammatory cytokine response of DCs after exposure to cell-free Tax have been reported. In this regard, a human cytokine protein array has been utilized for the detection of cytokines in supernatants of Tax-stimulated DCs. This array is composed of 16 wells and each well contains 16 different cytokine-specific antibodies spotted in triplicate. The multiplexed protein array-based analyses provide initial clues about the pro-inflammatory cytokines that are involved in the Tax-mediated effects on DCs and may provide insights into the mechanisms of HTLV-1-induced disease pathogenesis.

Section snippets

Generation of monocyte-derived dendritic cells (MDDCs)

MDDCs were generated from peripheral blood mononuclear cells (PBMCs) as described by Ahuja et al. [25]. Briefly, PBMCs were labeled with anti-CD14-coated magnetic beads (Miltenyi Biotec Inc., Auburn, CA) and monocytes were magnetically purified as described by the manufacturer (VarioMACS; Miltenyi Biotec). Purified monocytes were differentiated to immature DCs by culture in complete medium comprised of BioWhittaker™ RPMI-1640 with l-glutamine (Cambrex, Walkersville, MD) supplemented with 10%

HTLV-1 Tax induces the production of cytokines from primary human MDDCs

To examine whether extracellular Tax induces the production of cytokines from MDDCs, the optimal dose of protein essential to detect cytokine secretion was previously determined by exposing cells to an increasing concentration of Tax (Jain and Wigdahl, unpublished observations). As a result of these studies, a concentration of 1 μg/ml of Tax was sufficient to induce detectable cytokine secretion and this concentration of Tax was used for all subsequent experiments. Immature DCs were exposed to

Discussion

Recent studies showed a role for dendritic cells in a number of inflammatory and neurodegenerative conditions, including bacterial and parasitic infections [30], [31], amyotrophic lateral sclerosis [32], multiple sclerosis [33], and in the animal model of experimental autoimmune encephalitis [31]. DCs are of particular significance in the pathogenesis of HTLV-1-associated neurologic disease, because the development of HAM/TSP is associated with their rapid maturation [22]. Furthermore, HTLV-1

Acknowledgements

These studies were supported by the United States Public Health Service/National Institutes of Health Grant CA54559 awarded to B. Wigdahl.

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