Synthesis and Evaluation of diaryl ether modulators of the leukotriene A4 hydrolase aminopeptidase activity.

Activation of the aminopeptidase (AP) activity of leukotriene A4 hydrolase (LTA4H) presents a potential therapeutic strategy for resolving chronic inflammation. Previously, ARM1 and derivatives were found to activate the AP activity using the alanine-p-nitroanilide (Ala-pNA) as a reporter group in an enzyme kinetics assay. As an extension of this previous work, novel ARM1 derivatives were synthesized using a palladium-catalyzed Ullmann coupling reaction and screened using the same assay. Analogue 5, an aminopyrazole (AMP) analogue of ARM1, was found to be a potent AP activator with an AC50 of 0.12 µM. An X-ray crystal structure of LTA4H in complex with AMP was refined at 2.7 Å. Despite its AP activity with Ala-pNA substrate, AMP did not affect hydrolysis of the previously proposed natural ligand of LTA4H, Pro-Gly-Pro (PGP). This result highlights a discrepancy between the hydrolysis of more conveniently monitored chromogenic synthetic peptides typically employed in assays and endogenous peptides. The epoxide hydrolase (EH) activity of AMP was measured in vivo and the compound significantly reduced leukotriene B4 (LTB4) levels in a murine bacterial pneumonia model. However, AMP did not enhance survival in the murine pneumonia model over a 14-day period. A liver microsome stability assay showed metabolic stability of AMP. The results suggested that accelerated Ala-pNA cleavage is not sufficient for predicting therapeutic potential, even when the full mechanism of activation is known.

A protective role for B-1 cells and oxidation-specific epitope IgM in lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a morbid fibrotic lung disease with limited treatment options. The pathophysiology of IPF remains poorly understood, and elucidation of the cellular and molecular mechanisms of IPF pathogenesis is key to the development of new therapeutics. B-1 cells are an innate B cell population which play an important role linking innate and adaptive immunity. B-1 cells spontaneously secrete natural IgM and prevent inflammation in several disease states. One class of these IgM recognize oxidation-specific epitopes (OSE), which have been shown to be generated in lung injury and to promote fibrosis. A main B-1 cell reservoir is the pleural space, adjacent to the typical distribution of fibrosis in IPF. In this study, we demonstrate that B-1 cells are recruited to the lung during injury where they secrete IgM to OSE (IgM OSE ). We also show that the pleural B-1 cell reservoir responds to lung injury through regulation of the chemokine receptor CXCR4. Mechanistically we show that the transcription factor Id3 is a novel negative regulator of CXCR4 expression. Using mice with B-cell specific Id3 deficiency, a model of increased B-1b cells, we demonstrate decreased bleomycin-induced fibrosis compared to littermate controls. Furthermore, we show that mice deficient in secretory IgM ( sIgM -/- ) have higher mortality in response to bleomycin-induced lung injury, which is partially mitigated through airway delivery of the IgM OSE E06. Additionally, we provide insight into potential mechanisms of IgM in attenuation of fibrosis through RNA sequencing and pathway analysis, highlighting complement activation and extracellular matrix deposition as key differentially regulated pathways.

Hyperpolarized Xenon-129: A New Tool to Assess Pulmonary Physiology in Patients with Pulmonary Fibrosis.

PURPOSE: The existing tools to quantify lung function in interstitial lung diseases have significant limitations. Lung MRI imaging using inhaled hyperpolarized xenon-129 gas (129Xe) as a contrast agent is a new technology for measuring regional lung physiology. We sought to assess the utility of the 129Xe MRI in detecting impaired lung physiology in usual interstitial pneumonia (UIP). MATERIALS AND METHODS: After institutional review board approval and informed consent and in compliance with HIPAA regulations, we performed chest CT, pulmonary function tests (PFTs), and 129Xe MRI in 10 UIP subjects and 10 healthy controls. RESULTS: The 129Xe MRI detected highly heterogeneous abnormalities within individual UIP subjects as compared to controls. Subjects with UIP had markedly impaired ventilation (ventilation defect fraction: UIP: 30 ± 9%; healthy: 21 ± 9%; p = 0.026), a greater amount of 129Xe dissolved in the lung interstitium (tissue-to-gas ratio: UIP: 1.45 ± 0.35%; healthy: 1.10 ± 0.17%; p = 0.014), and impaired 129Xe diffusion into the blood (RBC-to-tissue ratio: UIP: 0.20 ± 0.06; healthy: 0.28 ± 0.05; p = 0.004). Most MRI variables had no correlation with the CT and PFT measurements. The elevated level of 129Xe dissolved in the lung interstitium, in particular, was detectable even in subjects with normal or mildly impaired PFTs, suggesting that this measurement may represent a new method for detecting early fibrosis. CONCLUSION: The hyperpolarized 129Xe MRI was highly sensitive to regional functional changes in subjects with UIP and may represent a new tool for understanding the pathophysiology, monitoring the progression, and assessing the effectiveness of treatment in UIP.

Substrate-dependent modulation of the leukotriene A4 hydrolase aminopeptidase activity and effect in a murine model of acute lung inflammation.

The aminopeptidase activity (AP) of the leukotriene A4 hydrolase (LTA4H) enzyme has emerged as a therapeutic target to modulate host immunity. Initial reports focused on the benefits of augmenting the LTA4H AP activity and clearing its putative pro-inflammatory substrate Pro-Gly-Pro (PGP). However, recent reports have introduced substantial complexity disconnecting the LTA4H modulator 4-methoxydiphenylmethane (4MDM) from PGP as follows: (1) 4MDM inhibits PGP hydrolysis and subsequently inhibition of LTA4H AP activity, and (2) 4MDM activates the same enzyme target in the presence of alternative substrates. Differential modulation of LTA4H by 4MDM was probed in a murine model of acute lung inflammation, which showed that 4MDM modulates the host neutrophilic response independent of clearing PGP. X-ray crystallography showed that 4MDM and PGP bind at the zinc binding pocket and no allosteric binding was observed. We then determined that 4MDM modulation is not dependent on the allosteric binding of the ligand, but on the N-terminal side chain of the peptide. In conclusion, our study revealed that a peptidase therapeutic target can interact with its substrate and ligand in complex biochemical mechanisms. This raises an important consideration when ligands are designed to explain some of the unpredictable outcomes observed in therapeutic discovery targeting LTA4H.

Utilizing flip angle/TR equivalence to reduce breath hold duration in hyperpolarized 129 Xe 1-point Dixon gas exchange imaging.

PURPOSE: To reduce scan duration in hyperpolarized 129 Xe 1-point Dixon gas exchange imaging by utilizing flip angle (FA)/TR equivalence. METHODS: Images were acquired in 12 subjects (n = 3 radiation therapy, n = 1 unexplained dyspnea, n = 8 healthy) using both standard (TR = 15 ms, FA = 20°, duration = 15 s, 998 projections) and "fast" (TR = 5.4 ms, FA = 12°, duration = 11.3 s, 2100 projections) acquisition parameters. For the fast acquisition, 3 image sets were reconstructed using subsets of 1900, 1500, and 1000 projections. From the resulting ventilation, tissue ("barrier"), and red blood cell (RBC) images, image metrics and biomarkers were compared to assess agreement between methods. RESULTS: Images acquired using both FA/TR settings had similar qualitative appearance. There were no significant differences in SNR, image mean, or image SD between images. Moreover, the percentage of the lungs in "defect", "normal", and "high" bins for each image (ventilation, RBC, barrier) was not significantly different among the acquisition types. After registration, comparison of 3D image metrics (Dice, volume similarity, average distance) agreed well between bins. Images using 1000 projections for reconstruction had no significant differences from images using all projections. CONCLUSION: Using flip angle/TR equivalence, hyperpolarized 129 Xe gas exchange images can be acquired via the 1-point Dixon technique in as little as 6 s, compared to ~15 s for previously reported parameter settings. The resulting images from this accelerated scan have no significant differences from the standard method in qualitative appearance or quantitative metrics.

Image- versus histogram-based considerations in semantic segmentation of pulmonary hyperpolarized gas images.

PURPOSE: To characterize the differences between histogram-based and image-based algorithms for segmentation of hyperpolarized gas lung images. METHODS: Four previously published histogram-based segmentation algorithms (ie, linear binning, hierarchical k-means, fuzzy spatial c-means, and a Gaussian mixture model with a Markov random field prior) and an image-based convolutional neural network were used to segment 2 simulated data sets derived from a public (n = 29 subjects) and a retrospective collection (n = 51 subjects) of hyperpolarized 129Xe gas lung images transformed by common MRI artifacts (noise and nonlinear intensity distortion). The resulting ventilation-based segmentations were used to assess algorithmic performance and characterize optimization domain differences in terms of measurement bias and precision. RESULTS: Although facilitating computational processing and providing discriminating clinically relevant measures of interest, histogram-based segmentation methods discard important contextual spatial information and are consequently less robust in terms of measurement precision in the presence of common MRI artifacts relative to the image-based convolutional neural network. CONCLUSIONS: Direct optimization within the image domain using convolutional neural networks leverages spatial information, which mitigates problematic issues associated with histogram-based approaches and suggests a preferred future research direction. Further, the entire processing and evaluation framework, including the newly reported deep learning functionality, is available as open source through the well-known Advanced Normalization Tools ecosystem.

Measures of ventilation heterogeneity mapped with hyperpolarized helium-3 MRI demonstrate a T2-high phenotype in asthma.

BACKGROUND: Hyperpolarized gas with helium (HHe-3) MR (magnetic resonance) is a noninvasive imaging method which maps and quantifies regions of ventilation heterogeneity (VH) in the lung. VH is an important feature of asthma, but little is known as to how VH informs patient phenotypes. PURPOSE: To determine if VH indicators quantified by HHe-3 MR imaging (MRI) predict phenotypic characteristics and map to regions of inflammation in children with problematic wheeze or asthma. METHODS: Sixty children with poorly-controlled wheeze or asthma underwent HHe-3 MRI, including 22 with bronchoalveolar lavage (BAL). The HHe-3 signal intensity defined four ventilation compartments. The non-ventilated and hypoventilated compartments divided by the total lung volume defined a VH index (VHI %). RESULTS: Children with VHI % in the upper quartile had significantly greater airflow limitation, bronchodilator responsiveness, blood eosinophils, expired nitric oxide (FeNO), and BAL eosinophilic or neutrophilic granulocyte patterns compared to children with VHI % in the lower quartile. Lavage return from hypoventilated bronchial segments had greater eosinophil % than from ventilated segments. CONCLUSION: In children with asthma, greater VHI % as measured by HHe-3 MRI identifies a severe phenotype with higher type 2 inflammatory markers, and maps to regions of lung eosinophilia. Listed on ClinicalTrials. gov (NCT02577497).

Clinical Impact of Multidisciplinary Outpatient Care on Outcomes of Patients with COPD.

Purpose: Heterogeneous nature of Chronic Obstructive Pulmonary Disease (COPD) must be comprehensively addressed. It is unclear if integrative multidisciplinary disease management (IMDM) can optimize clinical outcomes of patients with COPD. Methods: A single-center, retrospective cohort observational study with a historical intervention was conducted in a clinic specialized for COPD care. Patients with a confirmed diagnosis of COPD were administered IMDM with measurement of BODE score on initial and follow-up visits. Primary outcomes were dynamic changes in BODE quartiles after receiving IMDM. Results: Of 124 patients, 21% were misdiagnosed with COPD. Patients with a confirmed diagnosis of COPD were 50% female, median age 64 years (IQR 57-70), 43% actively smoking and initial visit median BODE quartile 2 (IQR 1-3). Three subgroups were identified based on the changes in BODE quartiles: worsened (21%), unchanged (55%) and improved (24%). At baseline, mMRC (median [IQR]) was higher in improved subgroup vs worsened and unchanged subgroup (3 [3, 4] vs 2 [1, 2] vs 2 [1, 3], p value 0.002) respectively. Drop in all components of BODE score was noted in worsened group, but significant improvement in mMRC with preservation of spirometry values was noted in the improved group. The incidence of smoking cigarettes changed from 39% to 26% during follow-up. Conclusion: Our study demonstrates that IMDM can be potentially effective in a subgroup of COPD patients. In others precipitous drop in lung function, activity tolerance, and subjective symptoms seems inevitable with worsening BODE quartiles.

Effect of Modifier Structure on the Activation of Leukotriene A4 Hydrolase Aminopeptidase Activity.

Activation of the leukotriene A4 hydrolase (LTA4H) aminopeptidase (AP) activity with 4-methoxydiphenylmethane (4MDM) promoted resolution of neutrophil infiltration in a murine cigarette smoke-induced model for emphysematous chronic obstructive pulmonary disease. Recently, 4-(4-benzylphenyl)thiazol-2-amine (ARM1) was published as a ligand for LTA4H with potential anti-inflammatory properties. To investigate the effect of modifier structure on enzyme kinetics of LTA4H, a series of analogues bearing structural features of ARM1 and 4MDM were synthesized using trifluoroborate Suzuki coupling reactions. Following, the 2.8 Å X-ray crystal structure of LTA4H complexed with 4-OMe-ARM1, a 4MDM-ARM1 hybrid molecule, was determined. Kinetic analysis showed that ARM1 and related analogues lowered affinity for the enzyme-substrate complex, resulting in a change of mechanism from hyperbolic mixed predominately catalytic activation (HMx(Sp < Ca)A) as observed for 4MDM to a predominately specific activation (HMx(Sp > Ca)A) mechanism. 4-OMe-ARM1 was then shown to dose responsively reduce LTB4 production in human neutrophils.

Suppression of aspirin-mediated eosinophil activation by prostaglandin E2: Relevance to aspirin and nonsteroidal anti-inflammatory drug hypersensitivity.

BACKGROUND: Aspirin-exacerbated respiratory disease (AERD) is characterized by severe, sometimes life-threatening reactions to nonsteroidal anti-inflammatory drugs (NSAIDs). Mechanisms driving the disease include overproduction of leukotrienes and loss of anti-inflammatory prostaglandin E2 (PGE2) production. Many cell types contribute to the disease; however, eosinophils are markedly elevated and are important drivers of pathologic findings. OBJECTIVE: To investigate the capacity of aspirin and NSAIDs to drive eosinophil activation and the ability of PGE2 to inhibit this activation. METHODS: Eosinophils were purified from blood of healthy individuals without AERD and stimulated with lysine aspirin, ketorolac, or sodium salicylate. The role of PGE2 in altering activation was determined by incubating eosinophils with increasing doses of PGE2 before lysine aspirin stimulation. Specific PGE2 receptor use was determined by incubating eosinophils with receptor agonists and antagonists before aspirin stimulation. Cysteinyl leukotrienes (CysLTs), leukotriene B4 (LTB4), and eosinophil-derived neurotoxin (EDN) were quantified by enzyme-linked immunosorbent assay. RESULTS: Stimulation of eosinophils with lysine aspirin, ketorolac, or sodium salicylate resulted in secretion of CysLTs and LTB4 in the absence of EDN release. Low doses of PGE2 inhibited LTB4 and CysLT release, an effect lost at higher PGE2 concentrations. Use of butaprost, an EP2 receptor agonist, suppressed lysine aspirin stimulation. This mechanism was supported by blocking activity of the EP1 and EP3 receptors. CONCLUSION: Eosinophils can be directly activated by NSAIDs via cyclooxygenase-independent pathways to produce CysLTs and LTB4. This effect can be inhibited by PGE2 acting through the EP2 receptor. The recognized loss of EP2 receptor expression combined with low PGE2 levels explains in part the sensitivity to NSAIDs.

Role of medical and molecular imaging in COPD.

Chronic obstructive pulmonary disease (COPD) is expected to climb on the podium of the leading causes of mortality worldwide in the upcoming decade. Clinical diagnosis of COPD has classically relied upon detecting irreversible airflow obstruction on pulmonary function testing as a global assessment of pulmonary physiology. However, the outcome is still not favorable to decrease mortality due to COPD. Progress made in both medical and molecular imaging fields are beginning to offer additional tools to address this clinical problem. This review aims to describe medical and molecular imaging modalities used to diagnose COPD and to select patients for appropriate treatments and to monitor response to therapy.

Leukotriene A4 Hydrolase Activation and Leukotriene B4 Production by Eosinophils in Severe Asthma.

Asthma is associated with the overproduction of leukotrienes (LTs), including LTB4. Patients with severe asthma can be highly responsive to 5-lipoxygenase (5-LO) inhibition, which blocks production of both the cysteinyl LTs and LTB4. Production of LTB4 has traditionally been ascribed to neutrophils, mononuclear phagocytes, and epithelial cells, and acts as a chemoattractant for inflammatory cells associated with asthma. The source of LTB4 is unclear, especially in eosinophilic asthma. We speculated that the benefit of 5-LO inhibition could be mediated in part by inhibition of eosinophil-derived LTB4. LTB4 concentrations were assayed in BAL fluid from patients with severe asthma characterized by isolated neutrophilic, eosinophilic, and paucigranulocytic inflammation. Expression of LTA4 hydrolase (LTA4H) by airway eosinophils was determined by immunohistochemistry (IHC). Subsequently, peripheral blood eosinophils were activated and secreted LTB4 was quantified by enzyme immunoassay. Blood eosinophil LTA4H expression was determined by flow cytometry, qPCR, and IHC. LTB4 concentrations were elevated in BAL fluid from patients with severe asthma, including those with isolated eosinophilic inflammation, and these eosinophils displayed LTA4H via IHC. LTA4H expression by blood eosinophils was confirmed by flow cytometry, IHC, and qPCR. Robust LTB4 production by blood eosinophils was observed in response to some, but not all, stimuli. We demonstrated that eosinophils express LTA4H transcripts and protein, and can be stimulated to secrete LTB4. We speculate that in many patients with asthma, eosinophil-derived LTB4 is increased, and this may contribute to the efficacy of 5-LO inhibition.

Validation of claims-based algorithms for pulmonary arterial hypertension.

Administrative claims studies do not adequately distinguish pulmonary arterial hypertension (PAH) from other forms of pulmonary hypertension (PH). Our aim is to develop and validate a set of algorithms using International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) codes and electronic medical records (EMR), to identify patients with PAH. From January 2012 to August 2015, the EMRs of patients with ICD-9-CM codes for PH with an outpatient visit at the University of Texas Medical Branch were reviewed. Patients were divided into PAH or non-PAH groups according to EMR encounter diagnosis. Patient demographics, echocardiography, right heart catheterization (RHC) results, and PAH-specific therapies were assessed. RHC measurements were reviewed to categorize cases as hemodynamically determined PAH or not PAH. Weighted sensitivity, specificity, and positive and negative predictive values were calculated for the developed algorithms. A logistic regression analysis was conducted to determine how well the algorithms performed. External validation was performed at the University of Virginia Health System. The cohort for the development algorithms consisted of 683 patients with PH, PAH group (n = 191) and non-PAH group (n = 492). A hemodynamic diagnosis of PAH determined by RHC was recorded in the PAH (26%) and non-PAH (3%) groups. The positive predictive value for the algorithm that included ICD-9-CM and PAH-specific medications was 66.9% and sensitivity was 28.2% with a c-statistic of 0.66. The positive predictive value for the EMR-based algorithm that included ICD-9-CM, EMR encounter diagnosis, echocardiography, RHC, and PAH-specific medication was 69.4% and a c-statistic of 0.87. A validation cohort of 177 patients with PH examined from August 2015 to August 2016 using EMR-based algorithms yielded a similar positive predictive value of 62.5%. In conclusion, claims-based algorithms that included ICD-9-CM codes, EMR encounter diagnosis, echocardiography, RHC, and PAH-specific medications better-identified patients with PAH than ICD-9-CM codes alone.

Macrophages redirect phagocytosis by non-professional phagocytes and influence inflammation.

Professional phagocytes (such as macrophages) and non-professional phagocytes (such as epithelial cells) clear billions of apoptotic cells and particles on a daily basis. Although professional and non-professional macrophages reside in proximity in most tissues, whether they communicate with each other during cell clearance, and how this might affect inflammation, is not known. Here we show that macrophages, through the release of a soluble growth factor and microvesicles, alter the type of particles engulfed by non-professional phagocytes and influence their inflammatory response. During phagocytosis of apoptotic cells or in response to inflammation-associated cytokines, macrophages released insulin-like growth factor 1 (IGF-1). The binding of IGF-1 to its receptor on non-professional phagocytes redirected their phagocytosis, such that uptake of larger apoptotic cells was reduced whereas engulfment of microvesicles was increased. IGF-1 did not alter engulfment by macrophages. Macrophages also released microvesicles, whose uptake by epithelial cells was enhanced by IGF-1 and led to decreased inflammatory responses by epithelial cells. Consistent with these observations, deletion of IGF-1 receptor in airway epithelial cells led to exacerbated lung inflammation after allergen exposure. These genetic and functional studies reveal that IGF-1- and microvesicle-dependent communication between macrophages and epithelial cells can critically influence the magnitude of tissue inflammation in vivo.

Ultrapressure liquid chromatography-tandem mass spectrometry assay using atmospheric pressure photoionization (UPLC-APPI-MS/MS) for quantification of 4-methoxydiphenylmethane in pharmacokinetic evaluation.

4-Methoxydiphenylmethane (4-MDM), a selective augmenter of Leukotriene A4 Hydrolase (LTA4H), is a new anti-inflammatory compound for potential treatment of chronic obstructive pulmonary disease (COPD). Currently, there is no liquid chromatography tandem mass spectrometric (LC-MS/MS) method for the quantification of 4-MDM. A major barrier for developing the LC-MS/MS method is the inability of electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) to ionize 4-MDM due to its hydrophobicity and lack of any functional group for ionization. With the advent of atmospheric pressure photoionization (APPI) technique, many hydrophobic compounds have been demonstrated to ionize by charge transfer reactions. In this study, a highly sensitive ultrapressure liquid chromatography tandem mass spectrometry assay using atmospheric pressure photoionization (UPLC-APPI-MS/MS) for the quantifications of 4-MDM in rat plasma has been developed and validated. 4-MDM was extracted from the plasma by solid phase extraction (SPE) and separated chromatographically using a reverse phase C8 column. The photoionization (PI) was achieved by introducing anisole as a dopant to promote the reaction of charge transfer. The assay with a linear range of 5 (LLOQ)-400ngmL(-1) met the regulatory requirements for accuracy, precision and stability. The validated assay was employed to quantify the plasma concentrations of 4-MDM after an oral dosing in Sprague Dawley (SD) rats.

Physical deconditioning as a cause of breathlessness among obese adolescents with a diagnosis of asthma.

BACKGROUND: Obese children frequently complain of breathlessness. Asthma and obesity can both contribute to the symptoms during exercise, and this symptom can contribute to a diagnosis of asthma in these children. Despite the high prevalence of obesity few studies have investigated the cardiopulmonary physiology of breathlessness in obese children with a diagnosis of asthma. METHODS: In this case-control study, thirty adolescents between age 12 and 19 were studied with baseline spirometry and a cardiopulmonary exercise test. Ten adolescents were normal controls, ten had obesity without a diagnosis of asthma, and ten had obesity with a history of physician-diagnosed asthma. RESULTS: Baseline characteristics including complete blood count and spirometry were comparable between obese adolescents with and without a diagnosis of asthma. During exercise, obese asthmatic and obese non-asthmatic adolescents had significantly reduced physical fitness compared to healthy controls as evidenced by decreased peak oxygen uptake after adjusting for actual body weight (21.7 ± 4.5 vs. 21.4 ± 5.4 vs. 35.3 ± 5.8 ml/kg/min, respectively). However, pulmonary capacity at the peak of exercise was comparable among all three groups as evidenced by similar pulmonary reserve. CONCLUSION: In this study, breathlessness was primarily due to cardiopulmonary deconditioning in the majority of obese adolescents with or without a diagnosis of asthma.

Apoptotic cell clearance by bronchial epithelial cells critically influences airway inflammation.

Lung epithelial cells can influence immune responses to airway allergens. Airway epithelial cells also undergo apoptosis after encountering environmental allergens; yet, relatively little is known about how these are cleared, and their effect on airway inflammation. Here we show that airway epithelial cells efficiently engulf apoptotic epithelial cells and secrete anti-inflammatory cytokines, dependent upon intracellular signalling by the small GTPase Rac1. Inducible deletion of Rac1 expression specifically in airway epithelial cells in a mouse model resulted in defective engulfment by epithelial cells and aberrant anti-inflammatory cytokine production. Intranasal priming and challenge of these mice with house dust mite extract or ovalbumin as allergens led to exacerbated inflammation, augmented Th2 cytokines and airway hyper-responsiveness, with decreased interleukin (IL)-10 in bronchial lavages. Rac1-deficient epithelial cells produced much higher IL-33 upon allergen or apoptotic cell encounter, with increased numbers of nuocyte-like cells. Administration of exogenous IL-10 'rescued' the airway inflammation phenotype in Rac1-deficient mice, with decreased IL-33. Collectively, these genetic and functional studies suggest a new role for Rac1-dependent engulfment by airway epithelial cells and in establishing the anti-inflammatory environment, and that defects in cell clearance in the airways could contribute to inflammatory responses towards common allergens.

A critical role for LTA4H in limiting chronic pulmonary neutrophilic inflammation.

Leukotriene A(4) hydrolase (LTA(4)H) is a proinflammatory enzyme that generates the inflammatory mediator leukotriene B(4) (LTB(4)). LTA(4)H also possesses aminopeptidase activity with unknown substrate and physiological importance; we identified the neutrophil chemoattractant proline-glycine-proline (PGP) as this physiological substrate. PGP is a biomarker for chronic obstructive pulmonary disease (COPD) and is implicated in neutrophil persistence in the lung. In acute neutrophil-driven inflammation, PGP was degraded by LTA(4)H, which facilitated the resolution of inflammation. In contrast, cigarette smoke, a major risk factor for the development of COPD, selectively inhibited LTA(4)H aminopeptidase activity, which led to the accumulation of PGP and neutrophils. These studies imply that therapeutic strategies inhibiting LTA(4)H to prevent LTB(4) generation may not reduce neutrophil recruitment because of elevated levels of PGP.

Role of 5-lipoxygenase in IL-13-induced pulmonary inflammation and remodeling.

Exaggerated levels of IL-13 and leukotriene (LT) pathway activation frequently coexist at sites of Th2 inflammation and in tissue fibrotic responses. However, the relationship(s) between the IL-13 and LTs in these responses have not been defined. We hypothesized that the 5-lipoxygenase (5-LO) pathway of LT metabolism plays an important role in the pathogenesis of IL-13-induced chronic inflammation and remodeling. To test this hypothesis, we evaluated the effects of IL-13 on components of the 5-LO metabolic and activation pathways. We also compared the effects of transgenic IL-13 in C57BL/6 mice with wild-type and null 5-LO genetic loci. These studies demonstrate that IL-13 increases the levels of mRNA encoding cytosolic phospholipase A(2), LTA(4) hydrolase, and 5-LO-activating protein without altering the expression of 5-LO, LTC(4) synthase, LTB(4) receptors 1 and 2, and cysteinyl-LT receptors 1 and 2. They also demonstrate that this activation is associated with the enhanced accumulation of LTB(4) but not of cysteinyl-LTs. Furthermore, they demonstrate that this stimulation plays a critical role in the pathogenesis of IL-13-induced inflammation, tissue fibrosis, and respiratory failure-induced death while inhibiting alveolar remodeling. Lastly, mechanistic insights are provided by demonstrating that IL-13-induced 5-LO activation is required for optimal stimulation and activation of TGF-beta(1) and the inhibition of matrix metalloproteinase-12. When viewed in combination, these studies demonstrate that 5-LO plays an important role in IL-13-induced inflammation and remodeling.