EDITORIALS association study of bronchodilator response in asthmatics. Pharmacogenomics J 2014;14:41–47. 14. Drake KA, Torgerson DG, Gignoux CR, Galanter JM, Roth LA, Huntsman S, Eng C, Oh SS, Yee SW, Lin L, et al. A genome-wide association study of bronchodilator response in Latinos implicates rare variants. J Allergy Clin Immunol 2014;133:370–378. 15. Israel E, Lasky-Su J, Markezich A, Damask A, Szefler SJ, Schuemann B, Klanderman B, Sylvia J, Kazani S, Wu R, et al.; SHARP Investigators. Genome-wide association study of short-acting
b2 -agonists: a novel genome-wide significant locus on chromosome 2 near ASB3. Am J Respir Crit Care Med 2015;191: 530–537. 16. The International HapMap Consortium. HapMap Genome Browser Release 28. International HapMap Project; 2014 [accessed 2014 Dec 5]. Available from: http://hapmap.ncbi.nlm.nih.gov/cgi-perl/gbrowse/ hapmap28_B36/
Copyright © 2015 by the American Thoracic Society
Cysteinyl Leukotrienes: An Innate System for Epithelial Control of Airway Smooth Muscle Proliferation? Cysteinyl leukotrienes (cysLTs) are products of arachidonic acid metabolism by the 5-lipoxygenase/leukotriene C4 synthase (5-LO/ LTC4S) synthetic pathway. LTC4, the parent cysLT, is rapidly synthesized in response to cellular activation, released by a specific transporter, and converted extracellularly to LTD4 and LTE4 through the sequential actions of extracellular enzymes (1). CysLTs are generated by activated mast cells, eosinophils, basophils, macrophages, and myeloid dendritic cells, all of which constitutively express the requisite enzymes necessary for the formation of LTC4. All are hematopoietic cellular constituents of allergic inflammation. CysLTs signal through at least three cognate receptors, termed the type 1 and type 2 cysLT receptors (CysLT1R and CysLT2R) (2, 3), and GPR99 (4). Because of their potent effects as smooth muscle spasmogens (5) (an effect attributed primarily to CysLT1R), and because of evidence that the production of cysLTs increased during spontaneous asthma exacerbations (6), cysLTs drew early interest as potential mediators of asthma. This led to the development of some of the first molecularly targeted drugs for asthma therapy, the 5lipoxygenase inhibitor, zileuton, and selective CysLT1R antagonists. Both classes of drugs show efficacy in improving baseline airflow measurements and reducing the frequency of asthma exacerbations (7, 8), supporting the pathobiologic role of the cysLTs in regulating bronchomotor tone in asthma. Advances in molecular biology have significantly expanded our understanding of the potential pathobiologic functions of cysLTs. In mouse models of allergic pulmonary inflammation, cysLTs play a critical role in the induction and amplification of Th2type immune responses. Mice lacking LTC4S or CysLT1R, with impaired cysLT generation or signaling, respectively, showed a marked reduction in the development of eosinophilic pulmonary inflammation and Th2 immunity in response to intranasal extracts from the house dust mites Dermatophagoides farinae or Dermatophagoides pteronyssinus (Dp) (9–11). CysLT generation in these studies conditioned dendritic cells for Th2 priming via an autocrine/paracrine action at CysLT1R. In another model, intranasal delivery of LTC4 to sensitized mice receiving ovalbumin challenges markedly amplified pulmonary eosinophil recruitment by a pathway involving the activation of CysLT2R on platelets (12). In a model of intranasal sensitization and challenge to the mold Alternaria alternata, the recruitment and activation of type 2 innate lymphoid cells depended on cysLTs (13). Last, in a model of chronic airway inflammation induced by repetitive ovalbumin challenges of sensitized mice, the administration of the CysLT1R 496
antagonist montelukast inhibited peribronchial collagen deposition and airway smooth muscle hyperplasia (14). Although the latter finding supports a potential direct role for cysLTs (and CysLT1R) in driving airway remodeling, the pleiotropic actions of cysLTs at multiple steps in the proximal innate immune response require that downstream effects on structural cells be interpreted with caution. In this issue of the Journal, Trian and colleagues (pp. 538–546) demonstrate a potential pathway by which cysLTs may elicit airway smooth muscle proliferation independent of the proximal immune response (15). They demonstrate that airway smooth muscle cells (ASM) cultured from bronchial biopsies of patients with severe asthma proliferated in response to cysLTs generated by Dp-stimulated airway epithelial cells. ASM from nonasthmatic controls did not respond to the epithelial supernatants and expressed substantially less CysLT1R mRNA and protein than did cells from severe asthmatics. The effect of Dp on epithelial cells was inhibited by antibodies against protease-activated receptor 2, suggesting Dp proteases were responsible for the activation of the 5-LO/LTC4S pathway in this system. Treatment of the epithelial cells with either zileuton or dexamethasone eliminated Dp induction of the growth factor activity and LTC4 synthesis. The epithelial cells showed substantial up-regulation of 5-LO, 5-LO-activating protein, and LTC4S mRNA transcript expression in response to Dp, suggesting epithelial cells (in contrast to hematopoietic cells) may require transcriptional induction to become competent to generate cysLTs at a level sufficient to cause physiologic effects on smooth muscle. The study by Trian and colleagues carries several potential implications and caveats. First, although hematopoietic cells almost certainly provide the majority of cysLTs under virtually all circumstances, the study suggests epithelial cells can inducibly express the 5-LO/LTC4S system and could contribute cysLT synthesis when perturbed by environmental danger signals such as proteases. This would be consistent with previous observations that bronchial epithelial cells up-regulate 5-LO, 5-LO–activating protein, and LTC4S expression in response to stimulation with bradykinin or lipopolysaccharide (16). Second, epithelially derived cysLTs elicited proliferation only in asthmatic ASM, but not control ASM, which was attributed to the markedly higher levels of CysLT1R expression by asthmatic ASM. This phenomenon has not been reported previously and seems at odds with the previous reports that patients with asthma and nonasthmatic controls exhibit equivalent sensitivity to bronchoconstriction elicited by inhalation challenges with LTD4, the preferred ligand for CysLT1R (17, 18). Because the ASM cells used in
American Journal of Respiratory and Critical Care Medicine Volume 191 Number 5 | March 1 2015
EDITORIALS the study by Trian and colleagues all came from donors with severe disease, it is not possible to determine whether CysLT1R expression by ASM varies with disease severity. The molecular basis for the up-regulated expression of CysLT1R in ASM from patients with severe asthma identified in this study could reflect an epigenetic change, as it is not only evident in cells obtained directly by microdissection but is also retained through multiple passages of culture. Finally, the proliferative action of cysLTs for ASM, which was identified in prior studies (19), suggests a potential application for CysLT1R antagonists in the prevention of airway remodeling. This possibility awaits a carefully done longitudinal study using CysLT1R antagonism with careful monitoring of physiologic and histologic endpoints. n Author disclosures are available with the text of this article at www.atsjournals.org. Joshua A. Boyce, M.D. Department of Medicine and Department of Pediatrics Harvard Medical School Boston, Massachusetts and Division of Rheumatology, Immunology and Allergy Brigham and Women’s Hospital Boston, Massachusetts Nora A. Barrett, M.D. Department of Medicine Harvard Medical School Boston, Massachusetts and Division of Rheumatology, Immunology and Allergy Brigham and Women’s Hospital Boston, Massachusetts
References 1. Austen KF, Maekawa A, Kanaoka Y, Boyce JA. The leukotriene E4 puzzle: finding the missing pieces and revealing the pathobiologic implications. J Allergy Clin Immunol 2009;124:406–414, quiz 415–416. 2. Lynch KR, O’Neill GP, Liu Q, Im DS, Sawyer N, Metters KM, Coulombe N, Abramovitz M, Figueroa DJ, Zeng Z, et al. Characterization of the human cysteinyl leukotriene CysLT1 receptor. Nature 1999;399:789–793. 3. Heise CE, O’Dowd BF, Figueroa DJ, Sawyer N, Nguyen T, Im DS, Stocco R, Bellefeuille JN, Abramovitz M, Cheng R, et al. Characterization of the human cysteinyl leukotriene 2 receptor. J Biol Chem 2000;275: 30531–30536. 4. Kanaoka Y, Maekawa A, Austen KF. Identification of GPR99 protein as a potential third cysteinyl leukotriene receptor with a preference for leukotriene E4 ligand. J Biol Chem 2013;288:10967–10972.
5. Griffin M, Weiss JW, Leitch AG, McFadden ER Jr, Corey EJ, Austen KF, Drazen JM. Effects of leukotriene D on the airways in asthma. N Engl J Med 1983;308:436–439. 6. Drazen JM, O’Brien J, Sparrow D, Weiss ST, Martins MA, Israel E, Fanta CH. Recovery of leukotriene E4 from the urine of patients with airway obstruction. Am Rev Respir Dis 1992;146:104–108. 7. Israel E, Cohn J, Dube´ L, Drazen JM; Zileuton Clinical Trial Group. Effect of treatment with zileuton, a 5-lipoxygenase inhibitor, in patients with asthma. A randomized controlled trial. JAMA 1996; 275:931–936. 8. Israel E, Chervinsky PS, Friedman B, Van Bavel J, Skalky CS, Ghannam AF, Bird SR, Edelman JM. Effects of montelukast and beclomethasone on airway function and asthma control. J Allergy Clin Immunol 2002;110:847–854. 9. Barrett NA, Maekawa A, Rahman OM, Austen KF, Kanaoka Y. Dectin-2 recognition of house dust mite triggers cysteinyl leukotriene generation by dendritic cells. J Immunol 2009;182:1119–1128. 10. Barrett NA, Rahman OM, Fernandez JM, Parsons MW, Xing W, Austen KF, Kanaoka Y. Dectin-2 mediates Th2 immunity through the generation of cysteinyl leukotrienes. J Exp Med 2011;208: 593–604. 11. Clarke DL, Davis NH, Campion CL, Foster ML, Heasman SC, Lewis AR, Anderson IK, Corkill DJ, Sleeman MA, May RD, et al. Dectin-2 sensing of house dust mite is critical for the initiation of airway inflammation. Mucosal Immunol 2014;7:558–567. 12. Cummings HE, Liu T, Feng C, Laidlaw TM, Conley PB, Kanaoka Y, Boyce JA. Cutting edge: Leukotriene C4 activates mouse platelets in plasma exclusively through the type 2 cysteinyl leukotriene receptor. J Immunol 2013;191:5807–5810. 13. Doherty TA, Khorram N, Lund S, Mehta AK, Croft M, Broide DH. Lung type 2 innate lymphoid cells express cysteinyl leukotriene receptor 1, which regulates TH2 cytokine production. J Allergy Clin Immunol 2013;132:205–213. 14. Henderson WR Jr, Chiang GK, Tien YT, Chi EY. Reversal of allergeninduced airway remodeling by CysLT1 receptor blockade. Am J Respir Crit Care Med 2006;173:718–728. 15. Trian T, Allard B, Dupin I, Carvalho G, Ousova O, Maurat E, Bataille J, Thumerel M, Begueret H, Girodet P-O, et al. House dust mites induce proliferation of severe asthmatic smooth muscle cells via an epithelium-dependent pathway. Am J Respir Crit Care Med 2015; 191:538–546. 16. Jame AJ, Lackie PM, Cazaly AM, Sayers I, Penrose JF, Holgate ST, Sampson AP. Human bronchial epithelial cells express an active and inducible biosynthetic pathway for leukotrienes B4 and C4. Clin Exp Allergy 2007;37:880–892. 17. Weiss JW, Drazen JM, McFadden ER Jr, Weller P, Corey EJ, Lewis RA, Austen KF. Airway constriction in normal humans produced by inhalation of leukotriene D. Potency, time course, and effect of aspirin therapy. JAMA 1983;249:2814–2817. 18. Weiss JW, Drazen JM, McFadden ER Jr, Weller PF, Corey EJ, Lewis RA, Austen KF. Comparative bronchoconstrictor effects of histamine, leukotriene C, and leukotriene D in normal human volunteers. Trans Assoc Am Physicians 1982;95:30–35. 19. Ravasi S, Citro S, Viviani B, Capra V, Rovati GE. CysLT1 receptorinduced human airway smooth muscle cells proliferation requires ROS generation, EGF receptor transactivation and ERK1/2 phosphorylation. Respir Res 2006;7:42.
Copyright © 2015 by the American Thoracic Society
The Promise and Peril of Functional Genomics in Chronic Obstructive Pulmonary Disease The advent of computed tomographic (CT) imaging has realized the promise of performing organ-specific assessments of acute and chronic medical conditions in vivo. The application of this imaging
Editorials
modality to characterizing the regional structural abnormalities present in chronic obstructive pulmonary disease (COPD) and other airway diseases has been well documented and evolving for
497
b2 -agonists: a novel genome-wide significant locus on chromosome 2 near ASB3. Am J Respir Crit Care Med 2015;191: 530–537. 16. The International HapMap Consortium. HapMap Genome Browser Release 28. International HapMap Project; 2014 [accessed 2014 Dec 5]. Available from: http://hapmap.ncbi.nlm.nih.gov/cgi-perl/gbrowse/ hapmap28_B36/
Copyright © 2015 by the American Thoracic Society
Cysteinyl Leukotrienes: An Innate System for Epithelial Control of Airway Smooth Muscle Proliferation? Cysteinyl leukotrienes (cysLTs) are products of arachidonic acid metabolism by the 5-lipoxygenase/leukotriene C4 synthase (5-LO/ LTC4S) synthetic pathway. LTC4, the parent cysLT, is rapidly synthesized in response to cellular activation, released by a specific transporter, and converted extracellularly to LTD4 and LTE4 through the sequential actions of extracellular enzymes (1). CysLTs are generated by activated mast cells, eosinophils, basophils, macrophages, and myeloid dendritic cells, all of which constitutively express the requisite enzymes necessary for the formation of LTC4. All are hematopoietic cellular constituents of allergic inflammation. CysLTs signal through at least three cognate receptors, termed the type 1 and type 2 cysLT receptors (CysLT1R and CysLT2R) (2, 3), and GPR99 (4). Because of their potent effects as smooth muscle spasmogens (5) (an effect attributed primarily to CysLT1R), and because of evidence that the production of cysLTs increased during spontaneous asthma exacerbations (6), cysLTs drew early interest as potential mediators of asthma. This led to the development of some of the first molecularly targeted drugs for asthma therapy, the 5lipoxygenase inhibitor, zileuton, and selective CysLT1R antagonists. Both classes of drugs show efficacy in improving baseline airflow measurements and reducing the frequency of asthma exacerbations (7, 8), supporting the pathobiologic role of the cysLTs in regulating bronchomotor tone in asthma. Advances in molecular biology have significantly expanded our understanding of the potential pathobiologic functions of cysLTs. In mouse models of allergic pulmonary inflammation, cysLTs play a critical role in the induction and amplification of Th2type immune responses. Mice lacking LTC4S or CysLT1R, with impaired cysLT generation or signaling, respectively, showed a marked reduction in the development of eosinophilic pulmonary inflammation and Th2 immunity in response to intranasal extracts from the house dust mites Dermatophagoides farinae or Dermatophagoides pteronyssinus (Dp) (9–11). CysLT generation in these studies conditioned dendritic cells for Th2 priming via an autocrine/paracrine action at CysLT1R. In another model, intranasal delivery of LTC4 to sensitized mice receiving ovalbumin challenges markedly amplified pulmonary eosinophil recruitment by a pathway involving the activation of CysLT2R on platelets (12). In a model of intranasal sensitization and challenge to the mold Alternaria alternata, the recruitment and activation of type 2 innate lymphoid cells depended on cysLTs (13). Last, in a model of chronic airway inflammation induced by repetitive ovalbumin challenges of sensitized mice, the administration of the CysLT1R 496
antagonist montelukast inhibited peribronchial collagen deposition and airway smooth muscle hyperplasia (14). Although the latter finding supports a potential direct role for cysLTs (and CysLT1R) in driving airway remodeling, the pleiotropic actions of cysLTs at multiple steps in the proximal innate immune response require that downstream effects on structural cells be interpreted with caution. In this issue of the Journal, Trian and colleagues (pp. 538–546) demonstrate a potential pathway by which cysLTs may elicit airway smooth muscle proliferation independent of the proximal immune response (15). They demonstrate that airway smooth muscle cells (ASM) cultured from bronchial biopsies of patients with severe asthma proliferated in response to cysLTs generated by Dp-stimulated airway epithelial cells. ASM from nonasthmatic controls did not respond to the epithelial supernatants and expressed substantially less CysLT1R mRNA and protein than did cells from severe asthmatics. The effect of Dp on epithelial cells was inhibited by antibodies against protease-activated receptor 2, suggesting Dp proteases were responsible for the activation of the 5-LO/LTC4S pathway in this system. Treatment of the epithelial cells with either zileuton or dexamethasone eliminated Dp induction of the growth factor activity and LTC4 synthesis. The epithelial cells showed substantial up-regulation of 5-LO, 5-LO-activating protein, and LTC4S mRNA transcript expression in response to Dp, suggesting epithelial cells (in contrast to hematopoietic cells) may require transcriptional induction to become competent to generate cysLTs at a level sufficient to cause physiologic effects on smooth muscle. The study by Trian and colleagues carries several potential implications and caveats. First, although hematopoietic cells almost certainly provide the majority of cysLTs under virtually all circumstances, the study suggests epithelial cells can inducibly express the 5-LO/LTC4S system and could contribute cysLT synthesis when perturbed by environmental danger signals such as proteases. This would be consistent with previous observations that bronchial epithelial cells up-regulate 5-LO, 5-LO–activating protein, and LTC4S expression in response to stimulation with bradykinin or lipopolysaccharide (16). Second, epithelially derived cysLTs elicited proliferation only in asthmatic ASM, but not control ASM, which was attributed to the markedly higher levels of CysLT1R expression by asthmatic ASM. This phenomenon has not been reported previously and seems at odds with the previous reports that patients with asthma and nonasthmatic controls exhibit equivalent sensitivity to bronchoconstriction elicited by inhalation challenges with LTD4, the preferred ligand for CysLT1R (17, 18). Because the ASM cells used in
American Journal of Respiratory and Critical Care Medicine Volume 191 Number 5 | March 1 2015
EDITORIALS the study by Trian and colleagues all came from donors with severe disease, it is not possible to determine whether CysLT1R expression by ASM varies with disease severity. The molecular basis for the up-regulated expression of CysLT1R in ASM from patients with severe asthma identified in this study could reflect an epigenetic change, as it is not only evident in cells obtained directly by microdissection but is also retained through multiple passages of culture. Finally, the proliferative action of cysLTs for ASM, which was identified in prior studies (19), suggests a potential application for CysLT1R antagonists in the prevention of airway remodeling. This possibility awaits a carefully done longitudinal study using CysLT1R antagonism with careful monitoring of physiologic and histologic endpoints. n Author disclosures are available with the text of this article at www.atsjournals.org. Joshua A. Boyce, M.D. Department of Medicine and Department of Pediatrics Harvard Medical School Boston, Massachusetts and Division of Rheumatology, Immunology and Allergy Brigham and Women’s Hospital Boston, Massachusetts Nora A. Barrett, M.D. Department of Medicine Harvard Medical School Boston, Massachusetts and Division of Rheumatology, Immunology and Allergy Brigham and Women’s Hospital Boston, Massachusetts
References 1. Austen KF, Maekawa A, Kanaoka Y, Boyce JA. The leukotriene E4 puzzle: finding the missing pieces and revealing the pathobiologic implications. J Allergy Clin Immunol 2009;124:406–414, quiz 415–416. 2. Lynch KR, O’Neill GP, Liu Q, Im DS, Sawyer N, Metters KM, Coulombe N, Abramovitz M, Figueroa DJ, Zeng Z, et al. Characterization of the human cysteinyl leukotriene CysLT1 receptor. Nature 1999;399:789–793. 3. Heise CE, O’Dowd BF, Figueroa DJ, Sawyer N, Nguyen T, Im DS, Stocco R, Bellefeuille JN, Abramovitz M, Cheng R, et al. Characterization of the human cysteinyl leukotriene 2 receptor. J Biol Chem 2000;275: 30531–30536. 4. Kanaoka Y, Maekawa A, Austen KF. Identification of GPR99 protein as a potential third cysteinyl leukotriene receptor with a preference for leukotriene E4 ligand. J Biol Chem 2013;288:10967–10972.
5. Griffin M, Weiss JW, Leitch AG, McFadden ER Jr, Corey EJ, Austen KF, Drazen JM. Effects of leukotriene D on the airways in asthma. N Engl J Med 1983;308:436–439. 6. Drazen JM, O’Brien J, Sparrow D, Weiss ST, Martins MA, Israel E, Fanta CH. Recovery of leukotriene E4 from the urine of patients with airway obstruction. Am Rev Respir Dis 1992;146:104–108. 7. Israel E, Cohn J, Dube´ L, Drazen JM; Zileuton Clinical Trial Group. Effect of treatment with zileuton, a 5-lipoxygenase inhibitor, in patients with asthma. A randomized controlled trial. JAMA 1996; 275:931–936. 8. Israel E, Chervinsky PS, Friedman B, Van Bavel J, Skalky CS, Ghannam AF, Bird SR, Edelman JM. Effects of montelukast and beclomethasone on airway function and asthma control. J Allergy Clin Immunol 2002;110:847–854. 9. Barrett NA, Maekawa A, Rahman OM, Austen KF, Kanaoka Y. Dectin-2 recognition of house dust mite triggers cysteinyl leukotriene generation by dendritic cells. J Immunol 2009;182:1119–1128. 10. Barrett NA, Rahman OM, Fernandez JM, Parsons MW, Xing W, Austen KF, Kanaoka Y. Dectin-2 mediates Th2 immunity through the generation of cysteinyl leukotrienes. J Exp Med 2011;208: 593–604. 11. Clarke DL, Davis NH, Campion CL, Foster ML, Heasman SC, Lewis AR, Anderson IK, Corkill DJ, Sleeman MA, May RD, et al. Dectin-2 sensing of house dust mite is critical for the initiation of airway inflammation. Mucosal Immunol 2014;7:558–567. 12. Cummings HE, Liu T, Feng C, Laidlaw TM, Conley PB, Kanaoka Y, Boyce JA. Cutting edge: Leukotriene C4 activates mouse platelets in plasma exclusively through the type 2 cysteinyl leukotriene receptor. J Immunol 2013;191:5807–5810. 13. Doherty TA, Khorram N, Lund S, Mehta AK, Croft M, Broide DH. Lung type 2 innate lymphoid cells express cysteinyl leukotriene receptor 1, which regulates TH2 cytokine production. J Allergy Clin Immunol 2013;132:205–213. 14. Henderson WR Jr, Chiang GK, Tien YT, Chi EY. Reversal of allergeninduced airway remodeling by CysLT1 receptor blockade. Am J Respir Crit Care Med 2006;173:718–728. 15. Trian T, Allard B, Dupin I, Carvalho G, Ousova O, Maurat E, Bataille J, Thumerel M, Begueret H, Girodet P-O, et al. House dust mites induce proliferation of severe asthmatic smooth muscle cells via an epithelium-dependent pathway. Am J Respir Crit Care Med 2015; 191:538–546. 16. Jame AJ, Lackie PM, Cazaly AM, Sayers I, Penrose JF, Holgate ST, Sampson AP. Human bronchial epithelial cells express an active and inducible biosynthetic pathway for leukotrienes B4 and C4. Clin Exp Allergy 2007;37:880–892. 17. Weiss JW, Drazen JM, McFadden ER Jr, Weller P, Corey EJ, Lewis RA, Austen KF. Airway constriction in normal humans produced by inhalation of leukotriene D. Potency, time course, and effect of aspirin therapy. JAMA 1983;249:2814–2817. 18. Weiss JW, Drazen JM, McFadden ER Jr, Weller PF, Corey EJ, Lewis RA, Austen KF. Comparative bronchoconstrictor effects of histamine, leukotriene C, and leukotriene D in normal human volunteers. Trans Assoc Am Physicians 1982;95:30–35. 19. Ravasi S, Citro S, Viviani B, Capra V, Rovati GE. CysLT1 receptorinduced human airway smooth muscle cells proliferation requires ROS generation, EGF receptor transactivation and ERK1/2 phosphorylation. Respir Res 2006;7:42.
Copyright © 2015 by the American Thoracic Society
The Promise and Peril of Functional Genomics in Chronic Obstructive Pulmonary Disease The advent of computed tomographic (CT) imaging has realized the promise of performing organ-specific assessments of acute and chronic medical conditions in vivo. The application of this imaging
Editorials
modality to characterizing the regional structural abnormalities present in chronic obstructive pulmonary disease (COPD) and other airway diseases has been well documented and evolving for
497
