Stimulation of toll-like receptor 2 with bleomycin results in cellular activation and secretion of pro-inflammatory cytokines and chemokines
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.
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Cell lines
The human monocytic cell line THP1 and the human embryonic kidney (HEK) 293 were obtained from American Type Culture Collection (ATCC Numbers TIB-202 and CRL-1573, respectively). THP1 cells express high levels of TLR2 mRNA (Fig. 1) (Razonable et al., 2005), and thus this cell line was selected to characterize the cytokine profile of TLR2-expressing cells. Indeed, THP1 has been used in many in vitro studies that have assessed cell activation during bleomycin exposure (Huang et al., 2002). THP1
Human monocytic cells secrete IL-1β, IL-8, and TNF-α during bleomycin exposure
The human monocytic cell line THP1 secretes the pro-inflammatory cytokines IL-1β and TNF-α and the chemokine IL-8 in a concentration-dependent manner during co-incubation with bleomycin (Fig. 2). The highest levels of IL-1β, IL-8, and TNF-α secretion were observed at 100 mU/ml—a concentration that was determined to be non-toxic to the monocytic cells (data not shown). At this stimulation concentration, the mean absolute level (±SD) of IL-1β was 250-fold higher (252.74 ± 51.29 pg/ml vs.
Discussion
This study demonstrates that the anti-neoplastic drug bleomycin induces cellular activation in human monocytic cells by a TLR2-dependent mechanism. The recognition of bleomycin as a pathogen-associated molecular pattern by TLR2 triggers the activation of intracellular signaling pathways that lead to the translocation of the transcription factor NF-κB, which in turn drives the upregulation of genes that encode for the chemokine IL-8 and the pro-inflammatory cytokines IL-1β and TNF-α. Hence, this
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