Leukotriene A4 Hydrolase: The Janus Enzyme Shows Its Ugly Side in Smokers Many uninvited immune cells ﬁnd their way into the lungs of smokers with chronic obstructive pulmonary disease (COPD). Although neutrophils and macrophages are among the ﬁrst cells to arrive in response to cigarette smoke, animal models of smoke-induced emphysema have conﬁrmed the pathogenic role of activated T and B cells, as well as antigen-presenting cells that act upstream of lymphocyte activation (1). Similarly, in smokers with emphysema, the acquired immune responses persist long after smoking cessation, and autoreactive T cells could predict loss of lung function (2). Although memory T and B cell responses are reactive to speciﬁc selfor neo- (modiﬁed-self) antigens, how innate immune cells, such as neutrophils that lack recall responses, continue their omnipresence in the lungs of former smokers with COPD has remained a mystery. ELR1 CXC chemokines (e.g., IL-8) bind to CXCR1 and CXCR2 receptors expressed on neutrophils, and attract them to sites of inﬂammation. Ensuing degradation of collagen by neutrophil-derived proteases (Figure 1) generates many tripeptide proline-glycine-proline (PGP) peptides that mimic key sequences found in ELR1 CXC chemokines, and act as a potent neutrophil chemoattractant (3). PGP can subsequently undergo N-terminal acetylation to produce AcPGP, which exhibits enhanced chemotactic activity, and both peptides have been implicated in the pathophysiology of COPD: signiﬁcant concentrations of PGP/AcPGP have been detected in bronchoalveolar lavage (BAL) ﬂuid, sputum, and plasma of patients with COPD, spiking immediately preceding an exacerbation (3–5); repeated intratracheal administration to mice of AcPGP alone drives emphysema (3); cigarette smoke exposure of mice drives AcPGP accumulation, and neutralization of this peptide ameliorates neutrophilic inﬂammation and emphysema (6). Extracellular leukotriene (LT) A4 hydrolase (LTA4H) degrades PGP and resolves neutrophilic inﬂammation (7), whereas intracellular epoxide hydrolase function of the same enzyme converts LTA4 to LTB4. LTB4 is a proinﬂammatory lipid mediator capable of recruiting and activating an array of cells, including neutrophils. Thus, LTA4H exhibits opposing pro- and anti-inﬂammatory roles that govern neutrophil recruitment. Although efﬁcient clearance of PGP occurs in acute neutrophilic inﬂammation, this system is perturbed by cigarette smoke in two ways. First, cigarette smoke chemically acetylates PGP, enhancing its chemotactic activity and protecting it from degradation by LTA4H. Second, biochemical and preliminary murine studies suggest that cigarette smoke can selectively abrogate the peptidase activity of LTA4H, with minimal effect on the hydrolase activity (7). Thus, cigarette smoke pushes LTA4H toward a uniquely proinﬂammatory phenotype, whereby LTB4 and PGP together induce increased pulmonary neutrophilic inﬂammation. In this issue of the Journal, the article by Wells and colleagues (pp. 51–61) extends these latter studies in a cigarette smoke–induced emphysema model and translates them into two human smoking/COPD cohorts (8). Cigarette smoke–exposed mice displayed elevated BAL ﬂuid concentrations of PGP and a profound loss of LTA4H peptidase activity, while maintaining
LTA4H hydrolase activity and LTB4 production. Consistently, healthy smokers displayed elevated LTA4H expression and LTB4 concentration, but with selective inhibition of the enzyme’s peptidase activity with ensuing PGP accumulation. This phenotype persisted in patients with COPD, and was intriguingly maintained despite smoking cessation—hinting at a mechanism that may underlie failure of neutrophilic inﬂammation to resolve. Although elevated concentrations of PGP have been previously reported in patients with COPD (4), it is noteworthy in this article that healthy smokers also displayed elevated PGP owing to reduced LTA4H peptidase activity. Concentrations of PGP in the BAL ﬂuid of healthy smokers and patients with COPD were found to be indistinguishable; however, AcPGP was only elevated in those with disease, potentially pointing to acetylation as a key process in disease progression. Despite diminished LTA4H peptidase activity persisting in ex-smokers with COPD AcPGP levels are seemingly reduced, most likely due to a reduction in PGP-generating enzymes. Given these ﬁndings, it will be intriguing to elucidate if infection could induce COPD exacerbation by driving a further spike in PGP-generating enzymes. The authors show that acrolein in cigarette smoke in part inhibits LTA4H peptidase activity. Thus, acrolein perturbs the PGP–LTA4H pathway in two ways: selective inhibition of the peptidase function presented here, and through the acetylation and protection of the peptide previously demonstrated (9). Acrolein is a component of cigarette smoke, but can also be generated physiologically during inﬂammation. A precedent exists for sulphydryl blocking agents selectively inhibiting the peptidase activity of LTA4H, and this could represent a means by which acrolein selectively abrogates this important function (10). Interestingly, acrolein scavengers, such as N-acetylcysteine and carbocysteine, have shown some clinical beneﬁt in COPD trials (11, 12), which could feasibly now be in part attributable to a reduction in PGP/AcPGP. There is now a compelling case for a role of PGP/AcPGP in the pathogenesis of COPD. The concept of a protease imbalance and matrikine-driven, self-sustaining cycle of inﬂammation in COPD progression is appealing, but does PGP instigate inﬂammation/ neutrophilia, or, rather, is it a biomarker of the disease? A study investigating the use of macrolides in COPD demonstrated amelioration of symptoms, and reduced neutrophilia and PGP concentration (5), but in which order do these events occur? Interestingly, neutralization of IL-8 and LTB4 in COPD sputum/BAL ﬂuid partially inhibits its capacity to drive neutrophilic inﬂammation, implying that there are other key mediators, of which PGP is an obvious candidate (13). If PGP is a key player in COPD development, then what is the best way to target it for pharmaceutical intervention? Targeting PGP-generating enzymes may be problematic, owing to the pleiotropic roles of matrix metalloproteinases and prolyl endopeptidase. Complementary peptides that neutralize PGP (arginine-threonine-arginine) (14) may offer therapeutic potential, as would CXCR1 and/or CXCR2 antagonists. The use of N-acetylcysteine or carbocysteine, which scavenge acrolein, may prove beneﬁcial either alone or in combination with a novel
Figure 1. Cigarette smoke disrupts leukotriene (LT) A4 hydrolase (LTA4H)–mediated resolution of pulmonary neutrophilic inflammation. (1) In response to infection or injury, resident cells within the lung will release chemoattractants that will promote neutrophil recruitment from the vasculature and into the tissue. Epithelial cells and alveolar macrophages, for example, may release IL-8 that will bind to CXCR1/2 on the surface of the neutrophil and promote recruitment. The intracellular activity of LTA4H within leukocytes can generate the lipid mediator LTB4 that can also promote neutrophil recruitment by binding to LTB4 receptor (BLT1). (2) Neutrophils release an array of proteases within the lung tissue—the coordinated action of matrix metalloproteinases (MMPs; especially MMP-1, -8, and -9) and prolyl endopeptidase released from the neutrophil targets extracellular matrix collagen, releasing the neutrophil chemoattractant, proline-glycine-proline (PGP). PGP binds CXCR1/2 on the neutrophil and sustains neutrophil recruitment. (3) To terminate PGP-directed neutrophilic inflammation, LTA4H is released into an extracellular environment to degrade the peptide. (4) Acrolein, derived from cigarette smoke or physiologically during inflammation (lipid peroxidation, metabolism of threonine or spermine), can inhibit LTA4H-mediated degradation of PGP, allowing the peptide to accumulate and maintain neutrophilic inflammation. (5) Acrolein (and other components of cigarette smoke) can also chemically acetylate PGP on its N terminus, completely protecting the peptide from LTA4H-mediated degradation, and thus facilitating neutrophil recruitment. AcPGP = acetylated PGP; PE = prolyl endopeptidase.
class of LTA4H peptidase activators (15). Furthermore, the studies described by Wells and colleagues highlight the risk of limiting PGP degradation by LTA4H, with clear repercussions for current therapeutic strategies that target LTA4H, given how such inhibitors may fail to distinguish between the opposing activities of the enzyme, and could inadvertently lead to persistent neutrophilia. Going forward, it will be important to more fully characterize and ascertain the signiﬁcance of the PGP pathway in these and other chronic neutrophilic diseases of the lung and extrapulmonary sites, as it is clearly one that represents an attractive, novel therapeutic strategy. n
Farrah Kheradmand, M.D. Department of Medicine and Department of Pathology & Immunology Baylor College of Medicine Houston, Texas and Michael E. DeBakey Department of Veterans Affairs Center for Translational Research on Inﬂammatory Diseases Houston, Texas
References Author disclosures are available with the text of this article at www.atsjournals.org. Robert Snelgrove, Ph.D. National Heart and Lung Institute Imperial College London London, United Kingdom
1. Kheradmand F, Shan M, Xu C, Corry DB. Autoimmunity in chronic obstructive pulmonary disease: clinical and experimental evidence. Expert Rev Clin Immunol 2012;8:285–292. 2. Xu C, Hesselbacher S, Tsai CL, Shan M, Spitz M, Scheurer M, Roberts L, Perusich S, Zarinkamar N, Coxson H, et al. Autoreactive t cells in human smokers is predictive of clinical outcome. Front Immunol 2012;3:267.
American Journal of Respiratory and Critical Care Medicine Volume 190 Number 1 | July 1 2014
EDITORIALS 3. Weathington NM, van Houwelingen AH, Noerager BD, Jackson PL, Kraneveld AD, Galin FS, Folkerts G, Nijkamp FP, Blalock JE. A novel peptide CXCR ligand derived from extracellular matrix degradation during airway inﬂammation. Nat Med 2006;12:317–323. 4. O’Reilly P, Jackson PL, Noerager B, Parker S, Dransﬁeld M, Gaggar A, Blalock JE. N-alpha-PGP and PGP, potential biomarkers and therapeutic targets for COPD. Respir Res 2009;10:38. 5. O’Reilly PJ, Jackson PL, Wells JM, Dransﬁeld MT, Scanlon PD, Blalock JE. Sputum PGP is reduced by azithromycin treatment in patients with COPD and correlates with exacerbations. BMJ Open 2013;3: e004140. 6. Braber S, Koelink PJ, Henricks PA, Jackson PL, Nijkamp FP, Garssen J, Kraneveld AD, Blalock JE, Folkerts G. Cigarette smoke–induced lung emphysema in mice is associated with prolyl endopeptidase, an enzyme involved in collagen breakdown. Am J Physiol Lung Cell Mol Physiol 2011;300:L255–L265. 7. Snelgrove RJ, Jackson PL, Hardison MT, Noerager BD, Kinloch A, Gaggar A, Shastry S, Rowe SM, Shim YM, Hussell T, et al. A critical role for LTA4H in limiting chronic pulmonary neutrophilic inﬂammation. Science 2010;330:90–94. 8. Wells JM, O’Reilly PJ, Szul T, Sullivan DI, Handley G, Garrett C, McNicholas CM, Roda MA, Miller BE, Tal-Singer R, et al. An aberrant leukotriene A4 hydrolase–proline-glycine-proline pathway in the pathogenesis of chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2014;190:51–61. 9. Hardison MT, Brown MD, Snelgrove RJ, Blalock JE, Jackson P. Cigarette smoke enhances chemotaxis via acetylation of prolineglycine-proline. Front Biosci (Elite Ed) 2012;4:2402–2409. 10. Orning L, Fitzpatrick FA. Modiﬁcation of leukotriene A(4) hydrolase/aminopeptidase by sulfhydryl-blocking reagents:
differential effects on dual enzyme activities by methylmethane thiosulfonate. Arch Biochem Biophys 1999;368: 131–138. 11. Zheng JP, Wen FQ, Bai CX, Wan HY, Kang J, Chen P, Yao WZ, Ma LJ, Li X, Raiteri L, et al.; PANTHEON study group. Twice daily N-acetylcysteine 600 mg for exacerbations of chronic obstructive pulmonary disease (PANTHEON): a randomised, double-blind placebo-controlled trial. Lancet Respir Med 2014;2: 187–194. 12. Zheng JP, Kang J, Huang SG, Chen P, Yao WZ, Yang L, Bai CX, Wang CZ, Wang C, Chen BY, et al. Effect of carbocisteine on acute exacerbation of chronic obstructive pulmonary disease (PEACE Study): a randomised placebo-controlled study. Lancet 2008;371: 2013–2018. 13. Beeh KM, Kornmann O, Buhl R, Culpitt SV, Giembycz MA, Barnes PJ. Neutrophil chemotactic activity of sputum from patients with COPD: role of interleukin 8 and leukotriene B4. Chest 2003;123: 1240–1247. 14. van Houwelingen AH, Weathington NM, Verweij V, Blalock JE, Nijkamp FP, Folkerts G. Induction of lung emphysema is prevented by L-arginine–threonine–arginine. FASEB J 2008;22: 3403–3408. 15. Paige M, Wang K, Burdick M, Park S, Cha J, Jeffery E, Sherman N, Shim YM. Role of leukotriene A 4 hydrolase aminopeptidase in the pathogenesis of emphysema. J Immunol 2014;192: 5059–5068.
Published 2014 by the American Thoracic Society
Untangling the Healthcare Use Patterns of Severe Sepsis Survivors Many factors are contributing to an increasing awareness of severe sepsis as a large healthcare problem in the United States and also worldwide. The unfortunate death of Rory Staunton in New York has forced a more public awareness of the diagnosis (1), while the Surviving Sepsis Campaign has emphasized healthcare worker awareness (2). As severe sepsis has far outstripped acute myocardial infarctions—in terms of incidence and cost (3)—and “septicemia” is the individual diagnosis that hospitals in the United States spend the most money on as a whole (4), emphasis now focuses on timely diagnosis of sepsis and improving acute care in the hospital (5). Nonetheless, the long-term effects of sepsis (or its care) on patients, families, and the healthcare system are not well understood. It’s important to ask, therefore, whether a diagnosis of severe sepsis on hospitalization alters the trajectory for patients after hospital discharge. In this issue of the Journal, Prescott and colleagues (pp. 62–69) report on the healthcare use of 1,083 elderly patients hospitalized with severe sepsis who survived to hospital discharge (6). The study linked two databases: the Health and Retirement Study containing detailed information on the individuals, such as functional status prior to and after hospitalization, and Medicare data, which allowed for identiﬁcation of patients with severe sepsis on hospitalization (from 1998 through 2005), and provided information on healthcare resource use. Comparisons of resource use were made to the same individual in the year prior to
hospitalization, and to hospitalized patients who did not have severe sepsis. This “double comparison” strengthens the study and allows us to draw conclusions regarding the trajectories of individuals with severe sepsis, and also to understand whether severe sepsis sets people apart from other hospitalized patients. The study suggests that hospitalization for severe sepsis represents a “bend in the curve” with regard to a patient’s health trajectory, because in the year after hospitalization, severe sepsis survivors spent fewer days at home (and more days in healthcare facilities) compared with the year prior. This ﬁnding supports growing evidence that any severe illness is associated with physical or emotional difﬁculties after hospital discharge (7), as well as the fact that survivors of severe sepsis are known to be at higher risk of death after hospitalization (8). The more complex analysis is the “difference-in-difference” comparison to a matched cohort of hospitalized patients without severe sepsis. This analysis allows us to understand whether the changes in healthcare use for severe sepsis survivors were similar or different to patients hospitalized for other reasons. First, the patients with severe sepsis had to be matched to control subjects without sepsis. This matching included whether or not a patient was admitted to an intensive care unit (ICU)—an important marker for general severity of illness. Less than 40% of the cohort with severe sepsis was admitted to an ICU during the hospitalization. This percentage is notable because while severe