Cancer Prevention Research Differential Modulation of Leukotriene B4 Synthesis and Degradation in Human Bronchoalveolar Lavage Cells by Lipopolysaccharide and Tobacco Smoke

Leukotrienes have been implicated to play a prominent inductive role in carcinogenesis. We previously reported that bronchoalveolar lavage (BAL) cells from smokers manifested higher levels of leukotriene B4 (LTB4) production than ex-smokers. This study aims to elucidate the underlying mechanism(s). BAL cells from current and former smokers were exposed to lipopolysaccharide (LPS) for up to 7 days. LPS induced the release of LTB4 from BAL cells and down-regulated 5-lipoxygenase (5-LOX) mRNA expression in a dose-dependent manner, followed by a decrease in 5-LOX protein production and normalization of LTB4 levels. Exogenous LTB4 inhibited LPS-induced 5-LOX activity and accentuated the downregulation of 5-LOX mRNA, whereas suppression of 5-LOX abrogated the LPS-induced changes, suggesting a negative feedback mechanism. LPS concomitantly induced expression and activity of the LTB4 metabolizing enzyme LTB4 ω-hydroxylase (LTB4OH) in exsmokers' BAL cells, but not in smokers' BAL cells. In vitro smoke exposure of ex-smokers' BAL cells also abrogated the LPS-induced up-regulation of LTB4OH mRNA expression. Furthermore, ex-smokers' BAL cells expressed significantly higher LTB4OHmRNA levels than smokers' BAL cells. Such differential modulation of LTB4 synthesis and degradation by LPS in the setting of tobacco smoke exposure suggests that mechanisms responsible for sustained elevation of LTB4 levels in the lung microenvironment may contribute to the pathogenesis of tobacco-related respiratory diseases such as lung cancer. By regulating the balance of LTB4 in the lung, LTB4OH may function as a suppressor of lung carcinogenesis. Arachidonic acid is converted within inflammatory cells to leukotrienes by lipoxygenases (LOX) and to prostaglandins by cyclooxygenases (COX; ref. 1). Leukotrienes are generated via the action of the 5-LOX enzyme in concert with the 5-LOX activating protein (FLAP; ref. 2). The biologically active 5-LOX pathway metabolites are leukotriene B4 (LTB4) and the cysteinyl leukotrienes (LTC4, LTD4, and LTE4). LTB4 is rapidly and specifically catabolized via ω-oxidation by LTB4 ω-hydroxylase (LTB4OH; ref. 3). LTB4OH belongs to a family of cytochrome P450 4F enzymes. This inactivation of endogenous LTB4 has been studied in various cellular systems including the human neutrophils, which efficiently metabolize LTB4 via a cytochrome P450 4F3–dependent pathway, to produce 20-hydroxy-LTB4 (refs. 4, 5). The 5-LOX pathway–derived leukotrienes have been extensively implicated in the pathogenesis of a variety of respiratory diseases, including allergic rhinitis, asthma, chronic obstructive pulmonary disease, pneumonia, scleroderma lung disease, acute lung injury, and lung malignancies (6–12). Alveolar macrophages are the predominant effector cells in the lung microenvironment and are a major source of LTB4, a potent leukocyte activator. LTB4 is a powerful chemotaxin, promoting recruitment and adhesion of leukocytes to vascular endothelium. It also elicits the production and release of proinflammatory cytokines from macrophages and lymphocytes. Leukotrienes may be involved in regulation of angiogenesis (13) and have been reported to play a significant role in the pathogenesis and progression of a variety of cancers, with 5-LOX expression being reported in lung, colon, breast, prostate, and other cancers (1, 7, 14–18). Lipopolysaccharide (LPS) is a glycolipid component of the Gram-negative bacterial cell wall. Among its myriad proinflammatory effects, it is known to modulate the production of arachidonic acid metabolites by monocytes and macrophages. LPS has been known to induce the synthesis of COX-derived prostaglandins (19, 20), whereas the role of LPS in modulating the 5-LOX pathway is less clear; prior work suggests that acute, high-dose LPS exposure primes for leukotriene release (21). Recent studies, however, indicate Authors' Affiliations: Division of Pulmonary and Critical Care Medicine, David Geffen School of Medicine at University of California at Los Angeles, Los Angeles, California and Scripps Green Hospital, La Jolla, California Received 01/06/2008; revised 03/03/2008; accepted 03/19/2008. Grant support: National Cancer Institute grants K23/CA88068 and U01/CA96134-01 (J.T. Mao) and Stop Cancer Miller Family Grant. Requests for reprints: Jenny T. Mao, Division of Pulmonary and Critical Care, CHS 37-131, David Geffen School of Medicine at University of California at Los Angeles, Los Angeles, CA 90095-1690. Phone: 310-825-3100; Fax: 310206-8622; E-mail: [email protected]. ©2008 American Association for Cancer Research. doi:10.1158/1940-6207.CAPR-08-0001 266 Cancer Prev Res 2008;1(4) September 2008 www.aacrjournals.org Cancer Research. on June 20, 2017. © 2008 American Association for cancerpreventionresearch.aacrjournals.org Downloaded from that this modulation is more complex than previously believed, as prolonged LPS exposure inhibits stimulated LTB4 release from rat alveolar macrophages (22) and stimulated cysteinyl leukotriene release from the monocyte-like cell line THP-1 (23). We have previously shown that LPS exposure (for 24 hours) significantly increases LTB4 production in ex vivo human bronchoalveolar lavage (BAL) cells obtained from both current and ex-smokers, with an enhanced responsiveness in BAL cells from smokers (24). Our study now explores the mechanisms underlying this important interaction between bacterial products, tobacco smoke, and leukotriene synthesis and degradation. We show that LPS induces the production of LTB4 by human BAL cells while simultaneously down-regulating the expression of 5-LOX mRNA in a timeand dose-dependent manner. We further show that inhibition of guanylyl cyclase abrogates the LPS-induced down-regulation of 5-LOX mRNA expression in BAL cells whereas activation of guanylyl cyclase accentuates the effect of LPS, suggesting the involvement of the cyclic GMP (cGMP) signaling pathway. In addition, LPS induced a concomitant increase in LTB4OH mRNA expression and enzymatic activity. However, the LPS-induced upregulation of LTB4OH mRNA expression was not observed in BAL cells obtained from current smokers. BAL cells from ex-smokers also had significantly higher level of constitutive LTB4OH mRNA expression than BAL cells from smokers. These findings suggest that tobacco smoke impairs the regulatory mechanisms responsible for maintaining the balance of LTB4 levels in the lung microenvironment both constitutively and in response to inflammatory stimuli such as LPS. Such differential patterns of expression and responsiveness may contribute to sustained elevation of LTB4 in the lungs of smokers and the pathogenesis of tobacco-related respiratory diseases such as chronic obstructive pulmonary disease and lung cancer. Materials and Methods