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Emphysema Aminopeptidase in the Pathogenesis of Hydrolase 4 Role

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Emphysema Aminopeptidase in the Pathogenesis of Hydrolase 4 Role The Journal of Immunology Role of Leukotriene A4 Hydrolase Aminopeptidase in the Pathogenesis of Emphysema Mikell Paige,* Kan Wang,† Marie Burdick,‡ Sunhye Park,‡ Josiah Cha,‡ Erin Jeffery,x Nicholas Sherman,x and Y. Michael Shim‡ The leukotriene A4 hydrolase (LTA4H) is a bifunctional enzyme with epoxy hydrolase and aminopeptidase activities. We hypoth- esize that the LTA4H aminopeptidase activity alleviates neutrophilic inflammation, which contributes to cigarette smoke (CS)– induced emphysema by clearing proline-glycine-proline (PGP), a triamino acid chemokine known to induce chemotaxis of neutrophils. To investigate the biological contributions made by the LTA4H aminopeptidase activity in CS-induced emphysema, we exposed wild-type mice to CS over 5 mo while treating them with a vehicle or a pharmaceutical agent (4MDM) that selectively augments the LTA4H aminopeptidase without affecting the bioproduction of leukotriene B4. Emphysematous phenotypes were assessed by premortem lung physiology with a small animal ventilator and by postmortem histologic morphometry. CS exposure acidified the airspaces and induced localization of the LTA4H protein into the nuclei of the epithelial cells. This resulted in accumulation of PGP in the airspaces by suppressing the LTA4H aminopeptidase activity. When the LTA4H aminopeptidase activity was selectively augmented by 4MDM, the levels of PGP in the bronchoalveolar lavage fluid and infiltration of neutrophils into the lungs were significantly reduced without affecting the levels of leukotriene B4. This protected murine lungs from CS- induced emphysematous alveolar remodeling. In conclusion, CS exposure promotes the development of CS-induced emphysema by suppressing the enzymatic activities of the LTA4H aminopeptidase in lung tissues and accumulating PGP and neutrophils in the airspaces. However, restoring the leukotriene A4 aminopeptidase activity with a pharmaceutical agent protected murine lungs from developing CS-induced emphysema. The Journal of Immunology, 2014, 192: 5059–5068. eukotriene A4 hydrolase (LTA4H) has been known as PGP, thus mitigating the influx of neutrophils into murine lungs a bifunctional enzyme. Although two enzymatic activities postinfluenza infection (20). L share an overlapping substrate site, their biological func- Pulmonary emphysema is a major manifestation of chronic tions are distinctive (1, 2). The LTA4H epoxy hydrolase (EH) obstructive pulmonary disease. It is characterized by alveolar de- converts leukotriene A4 (LTA4) to leukotriene B4 (LTB4), which is struction in patients due to infiltration of neutrophils, lymphocytes, a potent inducer of neutrophil, macrophage, and T lymphocyte and macrophages into cigarette smoke (CS)–exposed lungs (3, 8, 14, chemotaxis in human diseases (3–10). The LTA4H aminopepti- 21–23). Although a number of mechanisms were proposed to ex- dase degrades the N terminus of peptides. Several studies dem- plain the pathogenesis of emphysema, its molecular pathogenesis is onstrated that a chemotactic triamino acid peptide, proline- not yet clearly understood. Neutrophil-rich inflammation in em- glycine-proline (PGP), is produced due to breakdown of collagen physematous lungs of smokers led us to hypothesize that the LTA4H by prolyl-endopeptidase, and PGP has been shown to induce che- aminopeptidase activity and bioproduction/clearance of PGP motaxis of neutrophils by binding to CXCR2 (11–19). Recently, may play an important role during the development of CS-induced LTA4H aminopeptidase has been reported to break down and clear neutrophilic inflammation and emphysema. Our laboratory has previously reported that the LTA4H enzy- matic activities make important contribution to the development of emphysematous tissue alterations (3, 24, 25). LTA4H activity *Department of Chemistry and Biochemistry, George Mason University, Manassas, was found to influence severity of emphysematous alveolar remod- † VA 22030; Center for Drug Discovery, Georgetown University Medical Center, elinginmurinelungsexposedtotransgenicallyoverexpressedIL-13 Washington, DC 20057; ‡Division of Pulmonary and Critical Care Medicine, Depart- x ment of Medicine, University of Virginia, Charlottesville, VA 22908; and Depart- (3, 24). LTA4H EH activity was found to contribute to emphyse- ment of Microbiology, Immunology, and Cancer Biology, University of Virginia, matous alveolar remodeling and neutrophilic infiltration into lungs Charlottesville, VA 22908 postexposure of intranasal elastase (3, 24). Whereas a number of Received for publication February 18, 2014. Accepted for publication March 30, studies have characterized the importance of the LTA H EH, no 2014. 4 studies have investigated the biological contributions made by This work was supported by the Flight Attendant Medical Research Institute (to Y.M.S.), National Institutes of Health Grant K08HL91127 (to Y.M.S.), and an Ivy Foundation the LTA4H aminopeptidase during the development of emphy- Biomedical Research Grant (to Y.M.S.). sema. Therefore, we first investigated the CS-induced alterations Address correspondence and reprint requests to Dr. Y. Michael Shim, Division of in the localization and enzymatic activity of the LTA4H protein. Pulmonary and Critical Care Medicine, Department of Medicine, University of Virginia, These studies demonstrated that CS significantly increased the P.O. Box 800546, Charlottesville, VA 22908-0546. E-mail address: [email protected] amount of LTA4H protein in murine lungs and led to specific Abbreviations used in this article: AA, ambient air; BALF, bronchoalveolar lavage fluid; CDX, 2-hydroxypropyl-b-cyclodextrin; CS, cigarette smoke; EH, epoxy patterns of LTA4H protein localization in lung tissues. Chronic hydrolase; KO, knockout; Lm, mean cordlength; LTA4,leukotrieneA4; LTA4H, exposure to CS also caused acidification of the bronchoalveolar LTA4 hydrolase; LTB4, leukotriene B4; MMP, metalloproteinase; PGP, proline-glycine- lavage fluid (BALF) in mice, which suppressed the enzymatic proline; WT, wild-type. activity of the LTA4H aminopeptidase in the BALF. All of these Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 events promoted exaggerated bioproduction of PGP and LTB4. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1400452 5060 ROLE OF LEUKOTRIENE A4 HYDROLASE AMINOPEPTIDASE IN EMPHYSEMA When the activity of the LTA4H aminopeptidase was restored by (Promega) in PBS. The trachea was tied to keep the lungs inflated and then selectively augmenting it without changing the EH activity, murine fixed in 10 ml paraformaldehyde for 18 h. The fixed lungs were stained lungs were protected from CS-induced emphysematous damage due with H&E. Alveolar size was determined by mean cordlength (Lm), as previously described (3, 24, 25, 29, 30). Sequential digital pictures (at least to reduction in the levels of PGP and neutrophilic infiltration into 10 per animal) of the entire lungs were captured by an Axiostar micro- the lungs without changes in the levels of LTB4. These studies scope (Carl Zeiss Microimaging) and then processed by NIH Image 1.63 demonstrate that the LTA4H aminopeptidase pathway is an impor- on a Macintosh computer with a macro downloaded from the National tant contributor for the CS-induced neutrophilic inflammation, PGP Institutes of Health server user-macro directory (http://rsb.info.nih.gov/ nih-image/download/contrib/ChordLength.SurfaceArea). clearance, and emphysematous alveolar remodeling independent of the LTA4HEHpathway. Measurement of PGP in BALF Quantitative analysis of PGP in the BALF by HPLC-mass spectroscopy Materials and Methods proved to be highly unreliable presumably due to the matrix effects of the Formulation of 4MDM to improve water solubility and aromatic carbons generated from exposure to cigarette combustion. These characterization of 4MDM bioavailability aromatic carbons saturated the chromatography columns and made the columns unusable even after the first run of chromatography. Therefore, The solubility of 4MDM was enhanced by formulation with 2-hydroxy- a two-step preparation of the BALF was performed, as follows. First, the propyl-b-cyclodextrin (CDX) and dextrose. The vehicle was prepared as BALF was purified by normal flow HPLC. The retention time of a standard an aqueous solution of CDX and dextrose without 4MDM. Maximum solution of PGP was determined to guide fractionation of PGP in the tolerated dose was first determined with WT mice (n = 10), and then the BALF samples. Second, the levels of PGP were quantified using a Thermo CDX-4MDM dose equal to 25% of the maximum tolerated dose was used Electron TSQ Quantum Access MAX mass spectrometer system with a in all subsequent in vivo studies. The solutions were administered as Protana nanospray ion source interfaced to a custom-packed 8 cm 3 m m drinking water for mice in cages. The levels of 4MDM in BALF were 75- m (internal diameter) Phenomenex Jupiter 10 mC18 reverse-phase m quantified first by enriching 4MDM using C18 Sep-Pak (Waters) and then capillary column. A 0.5- L aliquot of each fractionated extract was measuring the levels by using HPLC equipped with a UV detector (HPLC- injected, and the samples were eluted from the column using a reverse- UV). The HPLC setup consisted of the following: Shimadzu CBM con- phase gradient from 0 to 100% acetonitrile at a flow rate of 0.5 mL/min troller, Shimadzu LC20AD pump, Shimadzu SPD20A UV detector, Shi- over 0.5 h. The nanospray ion source was operated at +3 kV. The multiple 2 madzu CTO20A column oven, Waters C18 Symmetry HPLC column, and reaction
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