The 5-Lipoxygenase Pathway
A New Therapeutic Target for the Treatment of Pulmonary Fibrosis

Darryl C. Zeldin, M.D.

National Institute of Environmental Health Sciences, NIH, Research Triangle Park, North Carolina

	ARTICLE

TOP ARTICLE REFERENCES

Fibrotic lung diseases are a diverse group of pulmonary disorders characterized by inflammation, mesenchymal cell proliferation, and deposition of extracellular matrix proteins, resulting in severe lung dysfunction. Despite advances in our understanding of the pathogenesis of these disorders at the cellular and molecular levels, the currently recommended treatments, corticosteroids, cytotoxic agents, or both, have limited clinical efficacy, and patients suffering from these disorders often progress to severe respiratory failure and death.

Arachidonic acid is a polyunsaturated fatty acid that can be metabolized by several enzymes to produce lipid mediators (eicosanoids) that affect lung function. The cyclooxygenases convert arachidonic acid to prostaglandin (PG) H₂, which is further metabolized to PGE₂, PGF₂, PGD₂, PGI₂, and thromboxane. One of these eicosanoids (PGE₂) has potent bronchodilatory, antiinflammatory, and antifibrotic effects in the lung via action on specific cell surface receptors (EP receptors). Indeed, mice genetically deficient in cyclooxygenase-1 have reduced PGE₂ in bronchoalveolar lavage fluid, enhanced bronchoconstriction, and increased lung inflammatory response to inhaled allergen (1). In addition, cyclooxygenase-2 null mice develop increased pulmonary fibrosis following administration of bleomycin or vanadium pentoxide (2, 3). Moreover, fibroblasts from patients with idiopathic pulmonary fibrosis have a diminished capacity to biosynthesize PGE₂ and an intrinsic defect in their ability to upregulate cyclooxygenase-2 in response to various stimuli (4). Together, these data suggest a protective role of cyclooxygenase-derived eicosanoids in general and PGE₂ in particular in the lung.

In contrast, 5-lipoxygenase metabolizes arachidonic acid to the labile 5-hydroperoxyeicosatetraenoic acid, which is converted to 5-hydroxyeicosatetraenoic acid and leukotriene A₄. Leukotriene A₄ is the precursor of leukotriene B₄ and the cysteinyl-leukotrienes (leukotriene C₄, leukotriene D₄, and leukotriene E₄), which possess bronchoconstrictive and proinflammatory effects in the lung via action on specific leukotriene receptors. Thus, mice deficient in 5-lipoxygenase exhibit reduced airway inflammation and methacholine responsiveness following allergen challenge (5) and drugs that inhibit the 5-lipoxygenase pathway (e.g., zileuton) or antagonize the cysteinyl-leukotriene receptors (e.g., zafirlukast, montelukast) are effective treatments for patients with asthma (6). Interestingly, patients with idiopathic pulmonary fibrosis have increased lung leukotriene B₄ and leukotriene C₄ levels, suggesting constitutive activation of the 5-lipoxygenase pathway in this disorder (7). The role of this pathway, however, in the pathogenesis of lung fibrosis has been enigmatic. In this issue (pp. 229-235) of the American Journal of Respiratory and Critical Care Medicine, Peters-Golden and coworkers (8) demonstrate that mice deficient in 5-lipoxygenase have reduced capacity to biosynthesize cysteinyl-leukotrienes, reduced lung inflammation (assessed histologically and quantified on cytospins from collagenase lung digests), and reduced lung fibrosis (assessed using Masson's trichrome stain and by measuring total lung hydroxyproline levels) following intratracheal administration of bleomycin (8). Moreover, the 5-lipoxygenase null mice have increased production of the antifibrotic cytokine interferon- and the antiinflammatory/antifibrotic eicosanoid PGE₂, suggesting that the 5-lipoxygenase pathway may influence the fibrotic response either directly via production of leukotrienes or indirectly via modulation of the biosynthesis of other "protective" mediators.

Several criteria must be fulfilled to identify a candidate mediator as causally related to the development of pulmonary fibrosis. First, the mediator must be produced during the disease. As mentioned, patients with idiopathic pulmonary fibrosis have increased lung leukotriene levels (7). Peters-Golden and coworkers observe cysteinyl-leukotriene overproduction as early as Day 1 following administration of bleomycin in wild-type mice, thus suggesting involvement of these mediators in the acute inflammatory phase of this model (8). Importantly, increased production of cysteinyl-leukotrienes persists well beyond the initial insult, reaching maximal levels 21 d after bleomycina time when fibrosis is also maximal. Second, the mediator must be capable of producing the disease. Currently, no in vivo studies have documented that lung-specific overexpression of 5-lipoxygenase or administration of leukotrienes (either systemically or intratracheally) results in lung fibrosis; however, leukotrienes have been shown to exert potent effects on fibroblast migration, proliferation, and production of extracellular matrix proteins in vitro, suggesting that they may also be capable of stimulating mesenchymal cells to grow and deposit collagen in vivo (9, 10). Third, interventions that reduce the levels of the mediator should protect against the development of lung fibrosis. Peters-Golden and coworkers demonstrate that 5-lipoxygenase disruption, which attenuates leukotriene production following administration of bleomycin, results in protection from pulmonary fibrosis in mice (8). Although the exact mechanisms for this protective effect remain unknown (i.e., a direct effect of reduced leukotrienes versus an indirect effect on PGE₂ or interferon- levels), the study contributes significantly to the existing literature and goes a long way toward fulfilling Koch's postulates with regard to products of the 5-lipoxygenase pathway playing a causative role in the pathogenesis of pulmonary fibrosis.

How might these findings be translated into novel therapeutic alternatives for patients with fibrotic lung disease? As mentioned by Peters-Golden and coworkers, a trial of the 5-lipoxygenase inhibitor zileuton in patients with pulmonary fibrosis is already underway at their institution. In this phase II trial, patients with a histologic diagnosis of usual interstitial pneumonitis, stratified to control for pretreatment severity of their disease, will be randomly assigned to receive either zileuton (600 mg four times daily) or azathioprine plus prednisone (standard therapy) for 6 mo. The primary end point will be the percentage reduction in leukotriene levels in bronchoalveolar lavage fluid from pretreatment values. Secondary end points will include measures of clinical efficacy (progression-free survival time, quality of life), biomarkers of fibrotic lung disease activity (e.g., PGE₂, interferon-, transforming growth factor-, tumor necrosis factor-), and adverse effects of the pharmacotherapy. As in the case of the 5-lipoxygenase null mice, zileuton targets not only the cysteinyl-leukotrienes but also other mediators arising from the 5-lipoxygenase pathway, including leukotriene B₄ and 5-hydroxyeicosatetraenoic acid. Importantly, both of these eicosanoids stimulate phagocytosis and improve pulmonary clearance of gram-negative bacteria, and hence may be involved in host defense against these pathogens (11, 12). Thus, selective targeting of the cysteinyl-leukotriene receptors with either zafirlukast or montelukast, drugs that are less likely to influence host defense, have not been associated with liver toxicity, and have been used extensively and safely in patients with asthma, may provide an alternative treatment approach to modulating this pathway in pulmonary fibrosis. Given the wealth of data supporting a protective role of cyclooxygenase-derived PGE₂ in fibrotic lung disease and the potential that increased levels of this eicosanoid may, at least in part, be responsible for the observed phenotype of the 5-lipoxygenase null mice, it seems reasonable to also consider a pharmacotherapy trial with a stable EP receptor analog in patients with pulmonary fibrosis. Finally, because the biology of airway and alveolar remodeling are likely similar, the results of the study by Peters-Golden and coworkers might be relevant to the pathogenesis and treatment of airway remodeling in asthma.

	References

TOP ARTICLE REFERENCES

1. Gavett SH, Madison SL, Chulada PC, Scarborough PE, Qu W, Boyle JE, Tiano HF, Lee CA, Langenbach R, Roggli VL, Zeldin DC. Allergic lung responses are increased in prostaglandin H synthase-deficient mice. J Clin Invest 1999; 104: 721-732 [Medline].

2. Keerthisingam CB, Jenkins RG, Harrison NK, Hernandez-Rodriguez NA, Booth H, Laurent GJ, Hart SL, Foster ML, McAnulty RJ. Cyclooxygenase-2 deficiency results in a loss of the anti-proliferative response to transforming growth factor-beta in human fibrotic lung fibroblasts and promotes bleomycin-induced pulmonary fibrosis in mice. Am J Pathol 2001; 158: 1411-1422 [Abstract/Free Full Text].

3. Rice A, Zhang P, Moomaw C, Morgan D, Langenbach R, Bradbury A, Chulada PC, Zeldin DC, Bonner JC. Differential pulmonary fibrotic responses in prostaglandin H synthase-deficient mice following metal-induced lung injury. Am J Respir Crit Care Med 2000; 161: A752 .

4. Wilborn J, Crofford LJ, Burdick MD, Kunkel SL, Strieter RM, Peters-Golden M. Cultured lung fibroblasts isolated from patients with idiopathic pulmonary fibrosis have a diminished capacity to synthesize prostaglandin E2 and to express cyclooxygenase-2. J Clin Invest 1995; 95: 1861-1868 .

5. Irvin CG, Tu YP, Sheller JR, Funk CD. 5-Lipoxygenase products are necessary for ovalbumin-induced airway responsiveness in mice. Am J Physiol 1997; 272: L1053-L1058 [Abstract/Free Full Text].

6. Drazen JM, Israel E, O'Byrne PM. Treatment of asthma with drugs modifying the leukotriene pathway. N Engl J Med 1999; 340: 197-206 [Free Full Text].

7. Wilborn J, Bailie M, Coffey M, Burdick M, Strieter R, Peters-Golden M. Constitutive activation of 5-lipoxygenase in the lungs of patients with idiopathic pulmonary fibrosis. J Clin Invest 1996; 97: 1827-1836 [Medline].

8. Peters-Golden M, Bailie M, Marshall T, Wilke C, Phan SH, Toews GB, Moore BB. Protection from pulmonary fibrosis in leukotriene-deficient mice. Am J Respir Crit Care Med 2002; 165: 229-235 [Abstract/Free Full Text].

9. Phan SH, McGarry BM, Loeffler KM, Kunkel SL. Binding of leuko- triene C4 to rat lung fibroblasts and stimulation of collagen synthesis in vitro. Biochemistry 1988; 27: 2846-2853 [Medline].

10. Baud L, Perez J, Denis M, Ardaillou R. Modulation of fibroblast proliferation by sulfidopeptide leukotrienes: effect of indomethacin. J Immunol 1987; 138: 1190-1195 [Abstract/Free Full Text].

11. Bailie MB, Standiford TJ, Laichalk LL, Coffey MJ, Strieter R, Peters-Golden M. Leukotriene-deficient mice manifest enhanced lethality from Klebsiella pneumonia in association with decreased alveolar macrophage phagocytic and bactericidal activities. J Immunol 1996; 157: 5221-5224 [Abstract].

12. Mancuso P, Standiford TJ, Marshall T, Peters-Golden M. 5-Lipoxygenase reaction products modulate alveolar macrophage phagocytosis of Klebsiella pneumoniae. Infect Immun 1998; 66: 5140-5146 [Abstract/Free Full Text].