Stimulation of toll-like receptor 2 with bleomycin results in cellular activation and secretion of pro-inflammatory cytokines and chemokines

Abstract

The clinical use of bleomycin results in systemic and pulmonary inflammatory syndromes that are mediated by the production of cytokines and chemokines. In this study, we demonstrate that cell activation is initiated upon the recognition of bleomycin as a pathogen-associated molecular pattern by toll-like receptor (TLR) 2. The THP1 human monocytic cell line, which constitutively expresses high levels of TLR2, secretes interleukin (IL)-1β, IL-8, and tumor necrosis factor (TNF)-α during bleomycin exposure. The TLR2-dependent nature of cell activation and cytokine secretion is supported by (1) the inability of TLR2-deficient human embryonic kidney (HEK) 293 cells to exhibit nuclear factor-kappa B (NF-κB) activation and secrete IL-8 in response to bleomycin; (2) the acquired ability of HEK293 to exhibit NF-κB activation and secrete IL-8 upon experimental expression of TLR2; and (3) the inhibition of cell activation in TLR2-expressing HEK293 and THP1 by anti-TLR2 monoclonal antibody. Collectively, these observations identify TLR2 activation as a critical event that triggers NF-κB activation and secretion of cytokines and chemokines during bleomycin exposure. Our in vitro findings could serve as a molecular mechanism underlying the pro-inflammatory toxicity associated with bleomycin. Whether bleomycin engages with other cellular receptors that results in activation of alternate signaling pathways and whether the TLR2-agonist activity of bleomycin contribute to its anti-neoplastic property deserve further study.

Introduction

Bleomycin, a glycopeptide–polyketide bacterial metabolite that induces sequence-selective oxidative damage of nucleic acids, is clinically used in combination with radiation therapy and other chemotherapeutic agents for the treatment of various solid tumors and hematologic malignancies (de Wit et al., 2001, Gonzalez-Vela et al., 1988, Herod et al., 2000, Martinelli et al., 2003, Tokuhashi et al., 1997). It is also used as sclerosant to prevent the re-accumulation of pleural fluid in patients with recurrent effusions (Martinez-Moragon et al., 1997, Ong et al., 2000). The clinical benefits of bleomycin, however, are limited by severe adverse reactions that include a systemic inflammatory response syndrome, which is manifested as fever and chills, and a pneumonitis syndrome, which could progress to end-stage pulmonary fibrosis (Sleijfer, 2001, Sleijfer et al., 1998).

A pro-inflammatory environment is central to the pathogenesis of the adverse reactions to bleomycin. Patients with sepsis-like manifestations during bleomycin therapy exhibit elevated systemic levels of tumor necrosis factor (TNF)-α (Sleijfer et al., 1998). Human and animal studies have likewise highlighted the central role for the dominantly pro-inflammatory cytokines in the pathogenesis of “bleomycin lung” (Huang et al., 2002, Matsuoka et al., 2002, Miyamoto et al., 2002, Ortiz et al., 1998, Scheule et al., 1992, Smith et al., 1998). In vitro, the exposure of human and murine cells to bleomycin results in the activation of signal transduction pathways through mitogen-activated protein kinases (MAPK) (Day et al., 2001, Matsuoka et al., 2002, Zhang et al., 2000), the activation of transcription factors nuclear factor-kappa B (NF-κB), activator protein-1 and activating transcription factor-2 (Zhang et al., 2000, Day et al., 2001), and the secretion of cytokines including TNF-α, interleukin (IL)-β, IL-8, monocyte chemoattractant protein-1, and transforming growth factor-β (Huang et al., 2002, Miyamoto et al., 2002, Phan et al., 1991, Scheule et al., 1992). Nonetheless, the molecular mechanism that initially triggers this cascade of cell activation events that result in gene upregulation and biologic expression of cytokines remains elusive.

Recently, we observed that cell activation during the use of certain drug therapies is triggered by the recognition of the drug as a pathogen-associated molecular pattern by toll-like receptors (TLR)—a family of transmembrane pattern recognition receptors expressed in immune cells that signals the presence of non-self antigens and pathogenic microorganisms. The 10 currently characterized human TLRs, designated TLR1-10, have divergent leucine-rich extracellular domains that allow each TLR to recognize a specific pathogen-associated molecular pattern. Subsequent to the interaction with its specific ligand, the conserved cytoplasmic Toll/interleukin-1 receptor domain orchestrates a series of activation, phosphorylation, and recruitment events among distinct and common pathways leading to transcription of genes and secretion of cytokines. In the TLR2-amphotericin B model, the recognition of amphotericin B by TLR2 induces the activation of signaling pathways that result in NF-κB translocation and TNF-α, IL-1β, IL-6, and IL-8 secretion (Razonable et al., 2005, Sau et al., 2003).

In this study, we tested the hypothesis that a TLR2-dependent mechanism could account for the pro-inflammatory response to bleomycin. Our hypothesis is based on the similarities between bleomycin and amphotericin B in terms of clinical toxicity (e.g., fever) and biologic expression (e.g., TNF-α, IL-1β, and IL-8) (Huang et al., 2002, Razonable et al., 2005, Sleijfer et al., 1998, Smith et al., 1998). Indeed, bleomycin is a fermentation product of Streptomyces verticillus (Shen et al., 2002) and thus, like the Streptomyces nodosus-derived amphotericin B (Razonable et al., 2005), it might well be recognized by TLR2. To prove our hypothesis, we performed a series of in vitro experiments that compared the degree of activation between TLR2-expressing and TLR2-deficient human cells. Our collective observations implicate TLR2 activation as one of the initial critical events that triggers the release of pro-inflammatory cytokine and chemokine secretion during bleomycin exposure.