Thioredoxin-1 Protects against Neutrophilic Inflammation and Emphysema Progression in a Mouse Model of Chronic Obstructive Pulmonary Disease Exacerbation

Exacerbations of chronic obstructive pulmonary disease (COPD) are characterized by acute enhancement of airway neutrophilic inflammation under oxidative stress and can be involved in emphysema progression. However, pharmacotherapy against the neutrophilic inflammation and emphysema progression associated with exacerbation has not been established. Thioredoxin-1 has anti-oxidative and anti-inflammatory properties and it can ameliorate neutrophilic inflammation through anti-chemotactic effects and prevent cigarette smoke (CS)-induced emphysema. We aimed to determine whether thioredoxin-1 can suppress neutrophilic inflammation and emphysema progression in a mouse model of COPD exacerbation and if so, to reveal the underlying mechanisms.  Mice were exposed to CS and then challenged with polyinosine-polycytidylic acid [], an agonist for virus-induced innate immunity. Airway neutrophilic inflammation, oxidative stress and lung apoptosis were enhanced in smoke-sensitive C57Bl/6, but not in smoke-resistant NZW mice. Exposure to CS and poly(I:C) challenge accelerated emphysema progression in C57Bl/6 mice. Thioredoxin-1 suppressed neutrophilic inflammation and emphysema progression.  caused early neutrophilic inflammation through keratinocyte-derived chemokine and granulocyte-macrophage colony-stimulating factor (GM-CSF) release in the lung exposed to CS. Late neutrophilic inflammation was caused by persistent GM-CSF release, which thioredoxin-1 ameliorated. Thioredoxin-1 enhanced pulmonary mRNA expression of MAP kinase phosphatase 1 (MKP-1), and the suppressive effects of thioredoxin-1 on prolonged GM-CSF release and late neutrophilic inflammation disappeared by inhibiting MKP-1.  Using a mouse model of COPD exacerbation, we demonstrated that thioredoxin-1 ameliorated neutrophilic inflammation by suppressing GM-CSF release, which prevented emphysema progression. Our findings deepen understanding of the mechanisms underlying the regulation of neutrophilic inflammation by thioredoxin-1 and indicate that thioredoxin-1 could have potential as a drug to counteract COPD exacerbation.

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title: Multifaceted mechanisms in COPD: inflammation, immunity, and tissue repair and destruction
abstract: Chronic obstructive pulmonary disease is a leading global cause of morbidity and mortality that is characterised by inexorable deterioration of small airways obstruction with emphysema associated with cellular inflammation and structural remodelling. Other features include apoptosis as well as proliferation of cells, and both tissue repair and lack of tissue repair. Metalloprotease release, together with that of apoptotic factors, may underlie the emphysema, and, conversely, fibrosis of the small airways may be accounted for by the effects of growth factor activation. In advanced disease, influential factors include the development of autoimmunity, with activation of dendritic cells and T-helper cells of both type 1 and 2, and the senescence response. An inability of macrophages to ingest apoptosed cells and bacteria may exacerbate inflammatory responses. Systemic inflammation with concomitant cardiovascular disease and metabolic syndrome may reflect the effect of cigarette smoke on nonpulmonary cells. Corticosteroid resistance may be secondary to oxidative stress mechanisms, such as inactivation of histone deacetylases. The mechanisms of chronic obstructive pulmonary disease may be heterogeneous, according to severity, and clinical phenotypes need to be correlated with cellular and pathological processes. Treatments may be targeted to patients with specific mechanisms.

title: Complexity of terminal airspace geometry assessed by lung computed tomography in normal subjects and patients with chronic obstructive pulmonary disease.
abstract: Increases in the low attenuation areas (LAA) of chest x-ray computed tomography images in patients with chronic obstructive pulmonary disease (COPD) have been reported to reflect the development of pathological emphysema. We examined the statistical properties of LAA clusters in COPD patients and in healthy subjects. In COPD patients, the percentage of the lung field occupied by LAAs (LAA%) ranged from 2.6 to 67.6. In contrast, LAA% was always <30% in healthy subjects. The cumulative size distribution of the LAA clusters followed a power law characterized by an exponent D. We show that D is a measure of the complexity of the terminal airspace geometry. The COPD patients with normal LAA% had significantly smaller D values than the healthy subjects, and the D values did not correlate with pulmonary function tests except for the diffusing capacity of the lung. We interpret these results by using a large elastic spring network model and find that the neighboring smaller LAA clusters tend to coalesce and form larger clusters as the weak elastic fibers separating them break under tension. This process leaves LAA% unchanged whereas it decreases the number of small clusters and increases the number of large clusters, which results in a reduction in D similar to that observed in early emphysema patients. These findings suggest that D is a sensitive and powerful parameter for the detection of the terminal airspace enlargement that occurs in early emphysema.

title: Computed tomographic measurements of airway dimensions and emphysema in smokers. Correlation with lung function.
abstract: Chronic obstructive pulmonary disease (COPD) is characterized by the presence of airflow obstruction caused by emphysema or airway narrowing, or both. Low attenuation areas (LAA) on computed tomography (CT) have been shown to represent macroscopic or microscopic emphysema, or both. However CT has not been used to quantify the airway abnormalities in smokers with or without airflow obstruction. In this study, we used CT to evaluate both emphysema and airway wall thickening in 114 smokers. The CT measurements revealed that a decreased FEV(1) (%predicted) is associated with an increase of airway wall area and an increase of emphysema. Although both airway wall thickening and emphysema (LAA) correlated with measurements of lung function, stepwise multiple regression analysis showed that the combination of airway and emphysema measurements improved the estimate of pulmonary function test abnormalities. We conclude that both CT measurements of airway dimensions and emphysema are useful and complementary in the evaluation of the lung of smokers.

title: CT scan findings of emphysema predict mortality in COPD.
abstract: Emphysematous change as assessed by CT imaging has been reported to correlate with COPD prognostic factors such as FEV(1) and diffusing capacity of the lung for carbon monoxide (Dlco). However, few studies have assessed the relationship between CT scan assessment and COPD mortality from mild to severe stages of the disease. In this study, we analyzed this relationship in patients with various stages of COPD.



Two hundred and fifty-one outpatients with stable COPD were included in the study. CT scan and pulmonary function tests were performed at study entry in a single institution. The percentage of low attenuation area was measured to quantitatively evaluate emphysematous change with a custom-made software. Prognostic data also were collected, and the median follow-up time was 8 years.



Of the 251 patients, 79 died, with 40 classified as respiratory deaths not involving lung cancer. Univariate Cox analysis revealed that emphysematous change as assessed by CT scan, lung function, age, or BMI were significantly correlated with mortality. Multivariate analysis revealed that emphysematous change as assessed by CT scan had the best association with mortality.



Emphysematous change as assessed by CT scan predicts respiratory mortality in outpatients with various stages of COPD.

title: Persistent airway inflammation and emphysema progression on CT scan in ex-smokers observed for 4 years.
abstract: Tobacco smoking is a principal cause of COPD-emphysema (COPD-E). Whether discontinuing smoking for at least 4 years halts airway inflammation and progression of COPD-E in prior smokers is unknown. In this study we investigated whether discontinuing smoking for approximately 4 years in ex-smokers with GOLD (Global Initiative for Chronic Lung Disease) stage IIb (moderately severe) COPD-E stopped airway inflammation (ie, sputum biomarkers) and halted the progression of COPD-E on chest CT scan.



Ten ex-smokers with COPD-E who had quit smoking underwent chest CT scans to document the extent of COPD-E, assessment of lung function (FEV(1) and diffusing capacity of lung for carbon monoxide), sputum induction for biomarkers of inflammation (measured by enzyme-linked immunosorbent assay), and blood cotinine levels at baseline and approximately 4 years later. Normal healthy subjects (n = 7) and normal current smokers with no CT scan evidence of COPD-E (n = 8) served as sputum biomarker comparison groups.



After approximately 4 years of not smoking (documented by cotinine levels), ex-smokers with COPD-E had persistent increased levels of mediators of inflammation in sputum (myeloperoxidase, leukotriene B4, IL-8, monocyte chemoattractant protein-1, matrix metalloprotease-9), which was associated with significant progression of COPD-E on chest CT scan.



Cessation of tobacco smoking in heavy smokers with moderately severe COPD-E is associated with evidence of persistent airway inflammation and progression of COPD-E on CT scan 4 years later. Discontinuing smoking may slow the rate of progression of moderate severity COPD-E, but it does not prevent persistent airway inflammation and significant progression of COPD-E on CT scan.

title: Corticosteroid resistance in chronic obstructive pulmonary disease: inactivation of histone deacetylase
abstract: By contrast with patients with asthma, those with chronic obstructive pulmonary disease (COPD) are poorly responsive to the anti-inflammatory actions of corticosteroids, and these drugs provide little clinical benefit. In both diseases, multiple inflammatory genes are activated, which results from acetylation of core histones around which DNA is wound. This acetylation opens up the chromatin structure allowing gene transcription and synthesis of inflammatory proteins to proceed. Corticosteroids recruit histone deacetylase 2 (HDAC2) to the actively transcribing gene, which reverses this process and switches off inflammatory gene transcription. We propose that in patients with COPD, HDAC2 function is impaired by cigarette smoking and oxidative stress, leading to a pronounced reduction in responsiveness to corticosteroids. Oxidative stress could generate peroxynitrite, which impairs HDAC2 activity through nitration of critical tyrosine residues. This hypothesis raises the possibility that novel therapeutic approaches might unlock this corticosteroid resistance, leading to more effective anti-inflammatory treatments for COPD and other severe inflammatory diseases.

title: Effects of inhaled and oral glucocorticoids on inflammatory indices in asthma and COPD.
abstract: The role of glucocorticoids in the treatment of chronic obstructive pulmonary disease (COPD) is controversial. We have previously described high numbers of neutrophils and high concentrations of the inflammatory cytokines interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-alpha), and of the cell activation markers eosinophil cationic protein (ECP), eosinophil peroxidase (EPO), myeloperoxidase (MPO), and human neutrophil lipocalin (HNL) in COPD patients as compared with controls, and have postulated that the cytokines TNF-alpha and IL-8 play a role in propagating the inflammatory response in COPD. We have now studied the effects of inhaled and oral glucocorticoids on these inflammatory indices in induced sputum. Initially, we studied the effect of a 2-wk course of inhaled budesonide (800 mg twice daily for 2 wk) in 13 patients with severe COPD (mean FDV1: 35% predicted). There was no clinical benefit in either lung function or symptom scores, and no significant change in the inflammatory indices as measured by total and differential cell counts and concentrations of TNF-alpha eosinophil activation markers ECP and EPO, and neutrophil activation markers MPO and HNL. Because the lack of anti-inflammatory effect might have been due to poor drug delivery as a result of severe airflow limitation, we undertook a study examining the antiinflammatory effect of oral prednisolone (30 mg daily for 2 wk) in patients with COPD and undertook the same measurements in 10 patients with atopic asthma. Sputum eosinophil numbers, ECP, and EPO were significantly reduced in the asthmatic patients but were not modified in COPD. This confirms the clinical impression that inhaled steroids have little antiinflammatory effect, at least in the short term in this group of patients, and suggests that the inflammatory process in COPD is resistant to the antiinflammatory effect of glucocorticoids.

title: Biopsy neutrophilia, neutrophil chemokine and receptor gene expression in severe exacerbations of chronic obstructive pulmonary disease.
abstract: We have applied immunohistology and in situ hybridization to bronchial biopsies of patients with chronic obstructive pulmonary disease (COPD) to examine neutrophil recruitment and to determine neutrophil chemoattractant and CXC receptor (CXCR) 1 and CXCR2 gene expression associated with acute severe exacerbations. Cells were counted in endobronchial biopsies of (1) patients with COPD intubated for exacerbations (E-COPD; n = 15), (2) those with COPD in a stable phase of their disease (S-COPD; n = 7), and (3) nonsmoker surgical control subjects intubated for a nonrespiratory surgical procedure (n = 15). In comparison with the nonrespiratory surgical procedure and S-COPD groups, neutrophilia and gene expression for epithelial-derived neutrophil attractant-78 (CXCL5), interleukin-8 (CXCL8), CXCR1, and CXCR2 were each upregulated in the E-COPD group (p < 0.01); compared with the S-COPD group, by 97-, 6-, 6-, 3-, and 7-fold, respectively (p < 0.01). In E-COPD, there was a significant positive association between the number of neutrophils and CXCR2 mRNA-positive cells (r = 0.79; p < 0.01) but not between the number of neutrophils and CXCR1 mRNA-positive cells. At the time of sampling of the mucosa, there was no association between neutrophil number and either the length of intubation or viral infection. Thus, in COPD, in addition to CXCL8 and CXCR1, CXCL5 and CXCR2 appear to play important roles in the airway neutrophilia characteristic of severe exacerbations.

title: MMP-9, TIMP-1 and inflammatory cells in sputum from COPD patients during exacerbation
abstract: BackgroundIrreversible airflow obstruction in Chronic Obstructive Pulmonary Disease (COPD) is thought to result from airway remodelling associated with aberrant inflammation. Patients who experience frequent episodes of acute deterioration in symptoms and lung function, termed exacerbations, experience a faster decline in their lung function, and thus over time greater disease severity However the mechanisms by which these episodes may contribute to decreased lung function are poorly understood.This study has prospectively examined changes in sputum levels of inflammatory cells, MMP-9 and TIMP-1 during exacerbations comparing with paired samples taken prior to exacerbation.MethodsNineteen COPD patients ((median, [IQR]) age 69 [63 to 74], forced expiratory volume in one second (FEV1) 1.0 [0.9 to1.2], FEV1% predicted 37.6 [27.3 to 46.2]) provided sputa at exacerbation. Of these, 12 were paired with a samples collected when the patient was stable, a median 4 months [2 to 8 months] beforehand.ResultsMMP-9 levels increased from 10.5 μg/g [1.2 to 21.1] prior to exacerbation to 17.1 μg/g [9.3 to 48.7] during exacerbation (P < 0.01). TIMP-1 levels decreased from 3.5 μg/g [0.6 to 7.8] to 1.5 μg/g [0.3 to 4.9] (P = 0.16). MMP-9/TIMP-1 Molar ratio significantly increased from 0.6 [0.2 to 1.1] to 3.6 [2.0 to 25.3] (P < 0.05). Neutrophil, eosinophil and lymphocyte counts all showed significant increase during exacerbation compared to before (P < 0.05). Macrophage numbers remained level. MMP-9 levels during exacerbation showed highly significant correlation with both neutrophil and lymphocyte counts (Rho = 0.7, P < 0.01).ConclusionDuring exacerbation, increased inflammatory burden coincides with an imbalance of the proteinase MMP-9 and its cognate inhibitor TIMP-1. This may suggest a pathway connecting frequent exacerbations with lung function decline.

title: Severe acute exacerbations and mortality in patients with chronic obstructive pulmonary disease
abstract: Patients with chronic obstructive pulmonary disease (COPD) often present with severe acute exacerbations requiring hospital treatment. However, little is known about the prognostic consequences of these exacerbations. A study was undertaken to investigate whether severe acute exacerbations of COPD exert a direct effect on mortality.  Multivariate techniques were used to analyse the prognostic influence of acute exacerbations of COPD treated in hospital (visits to the emergency service and admissions), patient age, smoking, body mass index, co-morbidity, long term oxygen therapy, forced spirometric parameters, and arterial blood gas tensions in a prospective cohort of 304 men with COPD followed up for 5 years. The mean (SD) age of the patients was 71 (9) years and forced expiratory volume in 1 second was 46 (17)%.  Only older age (hazard ratio (HR) 5.28, 95% CI 1.75 to 15.93), arterial carbon dioxide tension (HR 1.07, 95% CI 1.02 to 1.12), and acute exacerbations of COPD were found to be independent indicators of a poor prognosis. The patients with the greatest mortality risk were those with three or more acute COPD exacerbations (HR 4.13, 95% CI 1.80 to 9.41).  This study shows for the first time that severe acute exacerbations of COPD have an independent negative impact on patient prognosis. Mortality increases with the frequency of severe exacerbations, particularly if these require admission to hospital.

title: Relationship between exacerbation frequency and lung function decline in chronic obstructive pulmonary disease
abstract: Chronic obstructive pulmonary disease (COPD) is characterised by both an accelerated decline in lung function and periods of acute deterioration in symptoms termed exacerbations. The aim of this study was to investigate whether these are related.  Over 4 years, peak expiratory flow (PEF) and symptoms were measured at home daily by 109 patients with COPD (81 men; median (IQR) age 68.1 (63–74) years; arterial oxygen tension (Pao2) 9.00 (8.3–9.5) kPa, forced expiratory volume in 1 second (FEV1) 1.00 (0.7–1.3) l, forced vital capacity (FVC) 2.51 (1.9–3.0) l); of these, 32 (29 men) recorded daily FEV1. Exacerbations were identified from symptoms and the effect of frequent or infrequent exacerbations (> or < 2.92 per year) on lung function decline was examined using cross sectional, random effects models.  The 109 patients experienced 757 exacerbations. Patients with frequent exacerbations had a significantly faster decline in FEV1 and peak expiratory flow (PEF) of –40.1 ml/year (n=16) and –2.9 l/min/year (n=46) than infrequent exacerbators in whom FEV1 changed by –32.1 ml/year (n=16) and PEF by –0.7 l/min/year (n=63). Frequent exacerbators also had a greater decline in FEV1 if allowance was made for smoking status. Patients with frequent exacerbations were more often admitted to hospital with longer length of stay. Frequent exacerbations were a consistent feature within a patient, with their number positively correlated (between years 1 and 2, 2 and 3, 3 and 4).  These results suggest that the frequency of exacerbations contributes to long term decline in lung function of patients with moderate to severe COPD.

title: Impact of exacerbations on emphysema progression in chronic obstructive pulmonary disease.
abstract: Low-attenuation areas assessed by computed tomography reflect the extent of pathological emphysema and correlate with airflow limitation and mortality in patients with chronic obstructive pulmonary disease. The cumulative size distribution of low-attenuation area clusters follows a power law characterized by an exponent, D. The values of D reflect the complexity of the terminal airspace geometry and sensitively detect alveolar structural changes. Exacerbations of chronic obstructive pulmonary disease have a negative impact on lung function and prognosis. However, the impact on emphysema progression remains unclear.



We investigated the relationship between exacerbation and emphysema progression assessed by computed tomography in patients with chronic obstructive pulmonary disease.



Exacerbations were prospectively recorded for 2 years. Annual changes in computed tomography parameters of emphysema were compared between patients with and without a history of exacerbations.



In patients with exacerbations, increases in the percentage of low-attenuation areas and decreases in D were greater than in patients without exacerbations. To interpret these results, we established a novel simulation model and found that not only enlargement of preexisting low-attenuation areas but also coalescence of adjoining low-attenuation areas due to alveolar wall destruction caused emphysema progression in patients with exacerbations.



This is the first longitudinal study to demonstrate that exacerbations are involved in emphysema progression in patients with chronic obstructive pulmonary disease. Emphysema progression should be evaluated as part of the outcomes of exacerbations in the management of chronic obstructive pulmonary disease.

title: Controlled trial of oral prednisone in outpatients with acute COPD exacerbation.
abstract: Corticosteroids are often used in the outpatient treatment of acute exacerbation of chronic obstructive pulmonary disease (COPD). To date, there are few data documenting the benefit of this practice. The objective of this randomized, double-blind, placebo-controlled trial was to assess the efficacy of corticosteroids in the outpatient treatment of COPD exacerbations. Twenty-seven patients presenting with acute COPD exacerbation were studied. In addition to continuing their previous medications and increasing their use of beta-agonists, patients were randomized to receive a 9-d tapering dose of either oral prednisone or placebo. Treatment with prednisone rather than placebo resulted in a more rapid improvement in arterial PO2 (PaO2) (1.12 mm Hg/d versus -0.03 mm Hg/day; p = 0.002), alveolar-arterial oxygen gradient (A-aDO2) (-1.16 mm Hg/d versus -0.03 mm Hg/day; p = 0.04), FEV1 (0.05 L/d versus 0.00 L/d; p = 0.006), and peak expiratory flow (PEF) (0.15 L/s/d versus 0.04 L/s/d; p = 0.009). Prednisone also resulted in fewer treatment failures (p = 0.002) and in a trend toward more rapid improvement in dyspnea scale scores. Outpatient treatment of acute COPD exacerbation with prednisone accelerates recovery of PaO2, A-aDO2, FEV1, and PEF, reduces the treatment failure rate, and improves subjective dyspnea.

title: Oral corticosteroids in patients admitted to hospital with exacerbations of chronic obstructive pulmonary disease: a prospective randomised controlled trial
abstract: The role of oral corticosteroids in treating patients with exacerbations of chronic obstructive pulmonary disease (COPD) remains contentious. We assessed in a prospective, randomised, double-blind, placebo-controlled trial the effects of oral corticosteroid therapy in patients with exacerbations of COPD requiring hospital admission.



We recruited patients with non-acidotic exacerbations of COPD who were randomly assigned oral prednisolone 30 mg once daily (n=29) or identical placebo (n=27) for 14 days, in addition to standard treatment with nebulised bronchodilators, antibiotics, and oxygen. We did spirometry and recorded symptom scores daily in inpatients. Time to discharge and withdrawals were noted in each group. We recalled patients at 6 weeks to repeat spirometry and collect data on subsequent exacerbations and treatment. Hospital stay was analysed by intention to treat and forced expiratory volume in 1 s (FEV1) according to protocol.



FEV1 after bronchodilation increased more rapidly and to a greater extent in the corticosteroid-treated group: percentage predicted FEV1 after bronchodilation rose from 25.7% (95% CI 21.0-30.4) to 32.2% (27.3-27.1) in the placebo group (p<0.0001) compared with 28.2% (23.5-32.9) to 41.5% (35.8-47.2) in the corticosteroid-treated group (p<0.0001). Up to day 5 of hospital stay, FEV1 after bronchodilation increased by 90 mL daily (50.8-129.2) and by 30 mL daily (10.4-49.6) in the placebo group (p=0.039). Hospital stays were shorter in the corticosteroid-treated group. Groups did not differ at 6-week follow-up.



These data provide evidence to support the current practice of prescribing low-dose oral corticosteroids to all patients with non-acidotic exacerbations of COPD requiring hospital admission.

title: Redox regulation of cellular activation.
abstract: Growing evidence has indicated that cellular reduction/oxidation (redox) status regulates various aspects of cellular function. Oxidative stress can elicit positive responses such as cellular proliferation or activation, as well as negative responses such as growth inhibition or cell death. Cellular redox status is maintained by intracellular redox-regulating molecules, including thioredoxin (TRX). TRX is a small multifunctional protein that has a redox-active disulfide/dithiol within the conserved active site sequence: Cys-Gly-Pro-Cys. Adult T cell leukemia-derived factor (ADF), which we originally defined as an IL-2 receptor alpha-chain/Tac inducer produced by human T cell lymphotrophic virus-I (HTLV-I)-transformed T cells, has been identified as human TRX. TRX/ADF is a stress-inducible protein secreted from cells. TRX/ADF has both intracellular and extracellular functions as one of the key regulators of signaling in the cellular responses against various stresses. Extracellularly, TRX/ADF shows a cytoprotective activity against oxidative stress-induced apoptosis and a growth-promoting effect as an autocrine growth factor. Intracellularly, TRX/ADF is involved in the regulation of protein-protein or protein-nucleic acid interactions through the reduction/oxidation of protein cysteine residues. For example, TRX/ADF translocates from the cytosol into the nucleus by a variety of cellular stresses, to regulate the expression of various genes through the redox factor-1 (Ref-1)/APEX. Further studies to clarify the regulatory roles of TRX/ADF and its target molecules may elucidate the intracellular signaling pathways in the responses against various stresses. The concept of "redox regulation" is emerging as an understanding of the novel mechanisms in the pathogenesis of several disorders, including viral infections, immunodeficiency, malignant transformation, and degenerative disease.

title: ATL‐derived factor (ADF), an IL‐2 receptor/Tac inducer homologous to thioredoxin; possible involvement of dithiol‐reduction in the IL‐2 receptor induction.
abstract: HTLV‐I transformed T cells not only express a large number of interleukin‐2 receptors [IL‐2R/p55(Tac)], but also produce a factor named ATL‐derived factor (ADF) that augments the expression of IL‐2R/p55(Tac). Based on a partial N‐terminal amino acid sequence, complementary DNA (cDNA) clones for human and mouse ADF were isolated and sequenced. Recombinant ADF produced by COS‐7 monkey kidney cells showed IL‐2R/Tac inducing activity on YT cells, which are sensitive for ADF. ADF mRNA was strongly expressed in HTLV‐I(+) T cells lines, but not in inactivated cells (THP‐1, unstimulated PBMC). Furthermore, in normal human peripheral blood mononuclear cells, the expression of ADF mRNA was enhanced by mitogens or phorbol myristate acetate, suggesting a possible involvement of ADF in the lymphocyte activation. Homology analysis revealed an unexpected relationship between ADF and dithiol‐reducing enzyme, thioredoxin, involved in many important biological reactions such as the conversion of ribonucleotides into deoxyribonucleotides, or the stabilization of glucocorticoid receptors. The biological significance of the generation of a redox potential in lymphocyte activation, and the possible involvement of dithiol reduction in the induction of IL‐2R/Tac are discussed.

title: IL-2 receptor gene activation by ATL-derived factor (ADF).
abstract: The interaction of Interleukin 2 (IL-2) and its specific receptor, Interleukin 2 receptor (IL-2R) (1), plays an important role for the growth and activation of lymphoid cells. One of the unique features of lymphocytes in immune system is the selective proliferation of the clones specific for variety of the antigens. In mature T lymphocytes (T-cells) which express antigen-specific T-cell receptors, the cell growth driven by IL-2/IL-2-R interaction appears to be strictly regulated by the initiative signals elicited by the interaction between T-cell receptors and the corresponding antigens.

title: Circulating thioredoxin suppresses lipopolysaccharide-induced neutrophil chemotaxis
abstract: Thioredoxin (Trx), a redox enzyme with a conserved active site (Cys-32–Gly–Pro–Cys-35), is induced and secreted into circulation in response to inflammation. Studies here demonstrate that elevating Trx levels in circulation either by i.v. injection of recombinant Trx or stimulating Trx release in Trx-transgenic mice dramatically blocks lipopolysaccharide (LPS)-stimulated neutrophil migration in the murine air pouch chemotaxis model. Furthermore, we show that leukocyte recruitment induced by the murine chemokines KC/GROα, RANTES (regulated upon activation, normal T cell expressed and secreted), and monocyte chemoattractant protein-1 (MCP-1) is suppressed also in Trx-transgenic mice. Addressing the mechanism responsible for this suppression, we show that circulating Trx blocks (i) the LPS-stimulated in vitro activation of neutrophil p38 mitogen-activated protein kinase, (ii) the normal down-regulation of CD62L on neutrophils migrating into the LPS-stimulated air pouch, and (iii) the in vitro adhesion of LPS-activated neutrophils on endothelial cells. However, as we also show, Trx does not alter the expression of endothelial cell adhesion molecules (intercellular adhesion molecule-1, vascular cell adhesion molecule-1, CD62P, and CD62E) within 3 h. Collectively, these findings indicate that elevated levels of circulating Trx interfere with chemotaxis by acting directly on neutrophils. We discuss these findings in the context of recent studies reporting beneficial effects of acutely elevated Trx in ischemic injury and negative effects associated with chronically elevated Trx in HIV disease.

title: Thioredoxin-1 suppresses systemic inflammatory responses against cigarette smoking.
abstract: Thioredoxin-1 (TRX) is a small redox-active protein with antioxidative effects and redox-regulating functions. Cigarette smoking is a major etiological factor in the pathogenesis of a variety of diseases and recruits systemic immune and inflammatory responses. This report demonstrates that TRX attenuates the systemic inflammatory responses induced by cigarette smoking. The mRNA expressions of tumor necrosis factor alpha (TNF-alpha) and macrophage migration inhibitory factor (MIF) were suppressed in the spleen of TRX overexpressing transgenic mice (TRX-tg) exposed to cigarette smoking, compared with control C57BL/6 mice. In addition, protein carbonylation, a marker of cellular protein oxidation, was enhanced by cigarette smoking in the tissues of heart and liver in control mice more than in TRX-tg mice. These findings suggest that TRX may suppress the systemic inflammatory responses against cigarette smoking.

title: Mammalian thioredoxin is a direct inhibitor of apoptosis signal‐regulating kinase (ASK) 1
abstract: Apoptosis signal‐regulating kinase (ASK) 1 was recently identified as a mitogen‐activated protein (MAP) kinase kinase kinase which activates the c‐Jun N‐terminal kinase (JNK) and p38 MAP kinase pathways and is required for tumor necrosis factor (TNF)‐α‐induced apoptosis; however, the mechanism regulating ASK1 activity is unknown. Through genetic screening for ASK1‐binding proteins, thioredoxin (Trx), a reduction/oxidation (redox)‐regulatory protein thought to have anti‐apoptotic effects, was identified as an interacting partner of ASK1. Trx associated with the N‐terminal portion of ASK1 in vitro and in vivo. Expression of Trx inhibited ASK1 kinase activity and the subsequent ASK1‐dependent apoptosis. Treatment of cells with N‐acetyl‐L‐cysteine also inhibited serum withdrawal‐, TNF‐α‐ and hydrogen peroxide‐induced activation of ASK1 as well as apoptosis. The interaction between Trx and ASK1 was found to be highly dependent on the redox status of Trx. Moreover, inhibition of Trx resulted in activation of endogenous ASK1 activity, suggesting that Trx is a physiological inhibitor of ASK1. The evidence that Trx is a negative regulator of ASK1 suggests possible mechanisms for redox regulation of the apoptosis signal transduction pathway as well as the effects of antioxidants against cytokine‐ and stress‐induced apoptosis.

title: Thioredoxin-1 Ameliorates Cigarette Smoke-Induced Lung Inflammation and Emphysema in Mice
abstract: Cigarette smoking is associated with the development of inflammatory lung diseases representing major health problems world-wide. We hypothesized that the redox-regulating molecule thioredoxin-1 (TRX), which shows anti-inflammatory, antioxidative, and antiapoptotic effects, could be induced by cigarette smoke (CS) and contribute to protect against CS-induced inflammation and lung destruction. In an acute study, human TRX transgenic mice and C57BL6/J mice were exposed to mainstream CS for 3 days. In the lungs of CS-exposed mice, bronchial epithelial injury and bronchoalveolar lavage neutrophilia were observed. Oxidative stress and apoptosis were enhanced, and the expression of cytokines macrophage inflammatory protein-2 and tumor necrosis factor (TNF)-α was increased 15.3- and 2.4-fold, respectively. Compared with C57BL6/J mice, TRX-transgenic mice had significantly less inflammation, oxidative damage, and apoptosis, as well as decreased levels of matrix metalloprotease-12 mRNA and serum TNF-α. When recombinant human TRX (40 μg/body/day, 3 days) was injected i.p. into CS-exposed C57BL6/J mice, a significant effect to offer protection against CS-induced lung injury was observed through suppression of neutrophil influx. In the chronic study, TRX-transgenic mice and C57BL6/J mice were exposed to CS for 6 months. This chronic exposure caused pulmonary emphysema in C57BL6/J mice accompanying prominent infiltration of macrophages and neutrophils to lung. These pathological changes were significantly suppressed in TRX-transgenic mice. In conclusion, TRX induction ameliorated CS-induced lung inflammation and emphysema in mice. TRX-1 may therefore play a preventive or therapeutic role in lung inflammatory disorders such as chronic obstructive pulmonary disease.

title: Human thioredoxin-1 ameliorates experimental murine colitis in association with suppressed macrophage inhibitory factor production.
abstract: Thioredoxin-1 (TRX) is a small multifunctional protein with antioxidative and redox-regulating functions. In this study, we investigated the significance of TRX in patients with inflammatory bowel disease (IBD) and the ability and mechanism to ameliorate experimental colitis.



Serum TRX and macrophage migration inhibitory factor (MIF) levels were measured in patients with IBD. The effects of TRX were evaluated in a dextran sulfate sodium (DSS)-induced colitis model by comparing TRX-overexpressing transgenic (TRX-TG) and control mice. We further evaluated the effect of recombinant human TRX (rhTRX) administration on DSS-induced colitis and colonic inflammation of interleukin (IL)-10 knockout (IL-10 KO) mice. Colonic inflammation was examined clinically and histologically. Proinflammatory cytokine levels were examined in colonic tissues, and MIF levels were measured in colonic tissues and sera in mice. The effect of TRX on MIF production was also analyzed in vitro.



Serum TRX and MIF levels were significantly higher in patients with IBD than normal controls, and TRX levels correlated with disease activity. TRX significantly ameliorated DSS-induced colitis and colonic inflammation of IL-10 KO mice. Increase of tumor necrosis factor-alpha and interferon-gamma in colonic tissues was significantly suppressed in TRX-TG mice compared with wild-type mice. MIF levels in colonic tissues and sera were significantly lower in TRX-TG mice than in wild-type mice, irrespective of DSS administration. Anti-TRX treatment exacerbated DSS-induced colitis. In vitro studies demonstrated that rhTRX suppressed MIF production in human monocyte cells.



TRX might have a potential as a novel therapeutic agent for the treatment of IBD.

title: Recombinant human thioredoxin suppresses lipopolysaccharide-induced bronchoalveolar neutrophil infiltration in rat.
abstract: Human thioredoxin (TRX) is a multifunctional redox-active protein. We previously reported that the intraperitoneal administration of recombinant human thioredoxin (rhTRX) attenuates inflammatory cytokine- or bleomycin-induced lung injury in mice. In this study, the effect of rhTRX injected intravenously after lipopolysaccharide (LPS) injection was analyzed in rats. Rats were injected with LPS followed by treatment with rhTRX. Although the bolus injection exerted no protective effect, continuous intravenous administration of rhTRX significantly suppressed percentage number of neutrophils in bronchoalveolar lavage fluid. Histological examination also showed that rhTRX decreased neutrophil infiltration in the lung tissues. Administered rhTRX was mainly excreted into the urine and the tissue accumulation of rhTRX in the lung was marginal. LPS-induced oxidative stress in the lung was slight in this model. These results demonstrated that continuous intravenous administration of rhTRX suppresses LPS-induced bronchoalveolar neutrophil infiltration by an anti-chemotactic effect. Administration of rhTRX did not promote the tumor growth nor affect chemosensitivity in the xenotransplantation model, suggesting the safety of rhTRX therapy for cancer patients.

title: Detection of rhinovirus in induced sputum at exacerbation of chronic obstructive pulmonary disease
abstract: Common colds are associated with exacerbations of chronic obstructive pulmonary disease (COPD). However, the role of the common cold virus (human rhinovirus) in the production of symptoms and lower airway inflammation at COPD exacerbation is unknown. Thirty three patients with moderate‐to‐severe COPD were seen at baseline, when the number of chest infections in the previous year was noted, and acutely at COPD exacerbation. Within 48 h after the onset of the exacerbation and at baseline, nasal aspirates and induced sputum were taken for rhinovirus reverse transcriptase polymerase chain reaction (RT‐PCR) analysis and determination of cytokine levels. Symptoms, recorded on diary cards, were noted and forced expiratory volume in one second (FEV1) and forced vital capacity (FVC) measured. At exacerbation, mean FEV1 and FVC fell significantly from baseline (p<0.001). Ten of 43 exacerbations were associated with rhinovirus infection, detected in induced sputum. In four of these, nasophageal samples contained no detectable rhinovirus. All baseline samples were negative for rhinovirus. The simultaneous presence of increased nasal discharge/nasal congestion (in 26 of the 43 exacerbations) and increased sputum (29 exacerbations) was strongly associated with the presence of rhinovirus (odds ratio 6.15; p=0.036). Total symptom scores were greater for rhinovirus as compared to nonrhinovirus exacerbations (p=0.039). Median baseline sputum interleukin‐6 levels rose from 90.2 to 140.3 pg·mL‐1 at exacerbation (p=0.005); the change was greater in the presence of rhinovirus infection (p=0.008). Rhinovirus infection can be detected at chronic obstructive pulmonary disease exacerbation. This is associated with elevation of lower airway interleukin‐6 levels, which may mediate lower airway symptom expression during chronic obstructive pulmonary disease exacerbations.

title: Respiratory viruses in exacerbations of chronic obstructive pulmonary disease requiring hospitalisation: a case-control study
abstract: Acute exacerbations of chronic obstructive pulmonary disease (AE-COPD) are a common cause of hospital admission. Many exacerbations are believed to be due to upper and/or lower respiratory tract viral infections, but the incidence of these infections in patients with COPD is still undetermined.  Respiratory syncytial virus (RSV), influenza A and B, parainfluenza 3, and picornaviruses were detected by nested reverse transcription polymerase chain reaction (RT-PCR) in upper (nasal lavage) and lower respiratory tract specimens (induced sputum). In a 2:1 case-control set up, 85 hospitalised patients with AE-COPD and 42 patients with stable COPD admitted for other medical reasons were studied.  Respiratory viruses were found more often in sputum and nasal lavage of patients with AE-COPD (48/85, 56%) than in patients with stable COPD (8/42, 19%, p<0.01). The most common viruses were picornaviruses (21/59, 36%), influenza A (15/59, 25%), and RSV (13/59, 22%). When specimens were analysed separately, this difference was seen in induced sputum (exacerbation 40/85 (47%) v stable 4/42 (10%), p<0.01) but was not significant in nasal lavage (exacerbation 26/85 (31%) v stable 7/42 (17%), p=0.14). In patients with AE-COPD, fever was more frequent in those in whom viruses were detected (12/48, 25%) than in those in whom viruses were not detected (2/37, 5%, p=0.03).  Viral respiratory pathogens are found more often in respiratory specimens of hospitalised patients with AE-COPD than in control patients. Induced sputum detects respiratory viruses more frequently than nasal lavage in these patients. These data indicate that nasal lavage probably has no additional diagnostic value to induced sputum in cross-sectional studies on hospitalised patients with AE-COPD and that the role of viral infection in these patients is still underestimated.

title: Cigarette smoke impacts immune inflammatory responses to influenza in mice.
abstract: Studies have shown that cigarette smoke impacts respiratory host defense mechanisms; however, it is poorly understood how these smoke-induced changes impact the overall ability of the host to deal with pathogenic agents.



The objective of this study was to investigate the impact of mainstream cigarette smoke exposure on immune inflammatory responses and viral burden after respiratory infection with influenza A.



C57BL/6 mice were sham- or smoke-exposed for 3 to 5 mo and infected with either 2.5 x 10(3) pfu (low dose) or 2.5 x 10(5) pfu (high dose) influenza virus.



Although smoke exposure attenuated the airway's inflammatory response to low-dose infection, we observed increased inflammation in smoke-exposed compared with sham-exposed mice after infection with high-dose influenza, despite a similar rate of viral clearance. The heightened inflammatory response was associated with increased expression of tumor necrosis factor-alpha, interleukin-6, and type 1 IFN in the airway, and increased mortality. Importantly, smoke exposure did not interfere with the development of influenza-specific memory responses; sham- and smoke-exposed animals were equally protected upon viral rechallenge.



Our study suggests that, in mice, cigarette smoke affects primary antiviral immune-inflammatory responses, whereas secondary immune protection remains intact.

title: Cigarette smoke selectively enhances viral PAMP- and virus-induced pulmonary innate immune and remodeling responses in mice.
abstract: Viral infections have more severe consequences in patients who have been exposed to cigarette smoke (CS) than in those not exposed to CS. For example, in chronic obstructive pulmonary disease (COPD), viruses cause more severe disease exacerbation, heightened inflammation, and accelerated loss of lung function compared with other causes of disease exacerbation. Symptomatology and mortality in influenza-infected smokers is also enhanced. To test the hypothesis that these outcomes are caused by CS-induced alterations in innate immunity, we defined the effects of CS on pathogen-associated molecular pattern-induced (PAMP-induced) pulmonary inflammation and remodeling in mice. CS was found to enhance parenchymal and airway inflammation and apoptosis induced by the viral PAMP poly(I:C). CS and poly(I:C) also induced accelerated emphysema and airway fibrosis. The effects of a combination of CS and poly(I:C) were associated with early induction of type I IFN and IL-18, later induction of IL-12/IL-23 p40 and IFN-gamma, and the activation of double-stranded RNA-dependent protein kinase (PKR) and eukaryotic initiation factor-2alpha (eIF2alpha). Further analysis using mice lacking specific proteins indicated a role for TLR3-dependent and -independent pathways as well as a pathway or pathways that are dependent on mitochondrial antiviral signaling protein (MAVS), IL-18Ralpha, IFN-gamma, and PKR. Importantly, CS enhanced the effects of influenza but not other agonists of innate immunity in a similar fashion. These studies demonstrate that CS selectively augments the airway and alveolar inflammatory and remodeling responses induced in the murine lung by viral PAMPs and viruses.

title: Treating Viral Exacerbations of Chronic Obstructive Pulmonary Disease: Insights from a Mouse Model of Cigarette Smoke and H1N1 Influenza Infection
abstract: Chronic obstructive pulmonary disease is a progressive lung disease that is punctuated by periods of exacerbations (worsening of symptoms) that are attributable to viral infections. While rhinoviruses are most commonly isolated viruses during episodes of exacerbation, influenza viruses have the potential to become even more problematic with the increased likelihood of an epidemic.  This study examined the impact of current and potential pharmacological targets namely the systemic corticosteroid dexamethasone and the peroxisome proliferator-activated receptor- gamma agonist pioglitazone on the outcome of infection in smoke-exposed mice. C57BL/6 mice were exposed to room air or cigarette smoke for 4 days and subsequently inoculated with an H1N1 influenza A virus. Interventions were delivered daily during the course of infection. We show that smoke-exposed mice have an exacerbated inflammatory response following infection. While smoke exposure did not compromise viral clearance, precision cut lung slices from smoke-exposed mice showed greater expression of CC (MCP-1, -3), and CXC (KC, MIP-2, GCP-2) chemokines compared to controls when stimulated with a viral mimic or influenza A virus. While dexamethasone treatment partially attenuated the inflammatory response in the broncho-alveolar lavage of smoke-exposed, virally-infected animals, viral-induced neutrophilia was steroid insensitive. In contrast to controls, dexamethasone-treated smoke-exposed influenza-infected mice had a worsened health status. Pioglitazone treatment of virally-infected smoke-exposed mice proved more efficacious than the steroid intervention. Further mechanistic evaluation revealed that a deficiency in CCR2 did not improve the inflammatory outcome in smoke-exposed, virally-infected animals.  This animal model of cigarette smoke and H1N1 influenza infection demonstrates that smoke-exposed animals are differentially primed to respond to viral insult. While providing a platform to test pharmacological interventions, this model demonstrates that treating viral exacerbations with alternative anti-inflammatory drugs, such as PPAR-gamma agonists should be further explored since they showed greater efficacy than systemic corticosteroids.

title: Methodology for the measurement of mucociliary function in the mouse by scintigraphy.
abstract: The objective of the study was to develop a scintigraphic method for measurement of airway mucociliary clearance in small laboratory rodents such as the mouse. Previous investigations have characterized the secretory cell types present in the mouse airway, but analysis of the mucus transport system has been limited to in vitro examination of tissue explants or invasive in vivo measures of a single airway, the trachea. Three methods were used to deposit insoluble, radioisotopic colloidal particles: oropharyngeal aspiration, intratracheal instillation, and nose-only aerosol inhalation. The initial distribution of particles within the lower respiratory tract was visualized by gamma-camera, and clearance of particles was followed intermittently over 6 h and at the conclusion, 24 h postdelivery. Subsets of mice underwent lavage for evidence of tissue inflammation, and others were restudied for reproducibility of the methods. The aspiration and instillation methods of delivery led to greater distributions of deposited activity within the lungs, i.e., approximately 60--80% of the total respiratory tract radioactivity, whereas the nose-only aerosol technique attained a distribution of 32% to the lungs. However, the aerosol technique maximized the fraction of particles that cleared the airway over a 24-h period, i.e, deposited onto airway epithelial surfaces and cleared by mucociliary function such that lung retention at 24 h averaged 57% for delivery by aerosol inhalation and > or =80% for the aspiration or intratracheal instillation techniques. Particle delivery methods did not cause lung inflammation/injury with use of inflammatory cells and chemoattractant cytokines as criteria. Scintigraphy can discern particle deposition and clearance from the lower respiratory tract in the mouse, is noninvasive and reproducible, and includes the capability for restudy and lung lavage when time course or chronic treatments are being considered.

title: The role of MAP kinase phosphatase-1 in the protective mechanism of dexamethasone against endotoxemia.
abstract: We have previously shown that glucocorticoids induce the expression of MAP kinase phosphatase (Mkp)(a)-1 in innate immune cells. Since Mkp-1 is a critical negative regulator of the innate immune response, we hypothesize that Mkp-1 plays a significant role in the anti-inflammatory action of glucocorticoids. The specific aim of the present study is to understand the role of Mkp-1 in the anti-inflammatory function of glucocorticoids.



Wild-type and Mkp-1(-/-) mice were treated with different doses of dexamethasone and then challenged with different doses of lipopolysaccharide (LPS). The survival and blood cytokines were assessed. The effects of dexamethasone on cytokine production in wild-type and Mkp-1(-/-) primary macrophages ex vivo were also examined.



We found that dexamethasone induced the expression of Mkp-1 in vivo. Dexamethasone treatment completely protected wild-type mice from the mortality caused by a relatively high dose of LPS. However, dexamethasone treatment offered only a partial protection to Mkp-1(-/-) mice. Dexamethasone attenuated TNF-alpha production in both wild-type and Mkp-1(-/-) mice challenged with LPS, although TNF-alpha production in Mkp-1(-/-) mice was significantly more robust than that in wild-type mice. Dexamethasone pretreatment shortened the duration of p38 and JNK activation in LPS-stimulated wild-type macrophages, but had little effect on p38 or JNK activation in similarly treated Mkp-1(-/-) macrophages.



Our results indicate that the inhibition of p38 and JNK activities by glucocorticoids is mediated by enhanced Mkp-1 expression. These results demonstrate that dexamethasone exerts its anti-inflammatory effects through both Mkp-1-dependent and Mkp-1-indepent mechanisms.

title: Neurotropin demonstrates cytoprotective effects in lung cells through the induction of thioredoxin-1.
abstract: Neurotropin, a nonprotein extract from inflamed rabbit skin inoculated with vaccinia virus, is well known as an analgesic drug, but its cytoprotective effects have not been explored. Because infection by viruses, such as human T-cell leukemia virus type I and Epstein-Barr virus, induces expression of the redox-regulating molecule, thioredoxin (TRX), we hypothesized that neurotropin would also be capable of regulating the redox balance and could be applied for the therapeutics of lung diseases caused by oxidative stress, such as chronic obstructive pulmonary disease. Neurotropin enhanced mRNA expression of the redox-regulating molecules, glutathione peroxidase and catalase and, particularly, TRX, in human lung adenocarcinoma A549 cells. Neurotropin also increased the cellular TRX content and regulated TRX release from cells. The cytoprotective effects of neurotropin against hydrogen peroxide and cigarette smoke extracts was demonstrated by an attenuation of lactate dehydrogenase release from oxidant-exposed A549 cells and the inhibition of apoptosis. This cytoprotection was linked with reduced activity of intracellular oxidants. Furthermore, neurotropin enhanced TRX expression in mouse lungs and ameliorated cigarette smoke-induced lung injury in mice, suggesting that its cytoprotective effects in lung epithelial cells are mediated through the induction of redox-regulating molecules that reduce intracellular oxidative activity.

title: The development of emphysema in cigarette smoke-exposed mice is strain dependent.
abstract: Only 20% of smokers develop chronic obstructive pulmonary disease. An important determinant of susceptibility is genomic variation. We undertook this study to define strains of mice with different susceptibilities for the development of smoking-induced emphysema because they could help identify genetic factors of susceptibility. NZWLac/J, C57BL6/J, A/J, SJ/L, and AKR/J strains were exposed to cigarette smoke for 6 months. Elastance (Htis), the extent of emphysema (mean linear intercept [Lm]), and the inflammatory cell and cytokine response were measured. NZWLac/J had no change in Lm or Htis (resistant). C57BL6/J, A/J, and SJ/L increased Lm, but not Htis (mildly susceptible). AKR/J increased Lm and Htis (super-susceptible). Only AKR/J had significant inflammation comprising macrophages, neutrophils, and T cells. The AKR/J showed an upregulation of Th1 cytokines whereas in the C57BL/6/J and NZWlac/J, cytokines did not change or were downregulated. We conclude that Lm, elastance, and inflammation are features that are needed to phenotype emphysema in mice. The inflammatory cell and cytokine profile may be an important determinant of the phenotype in response to cigarette smoke exposure. The identification of resistant and susceptible strains for the development of emphysema could be useful for genomic studies of emphysema susceptibility in mice and eventually in humans.

title: Tissue heterogeneity in the mouse lung: effects of elastase treatment.
abstract: We developed a network model in an attempt to characterize heterogeneity of tissue elasticity of the lung. The model includes a parallel set of pathways, each consisting of an airway resistance, an airway inertance, and a tissue element connected in series. The airway resistance, airway inertance, and the hysteresivity of the tissue elements were the same in each pathway, whereas the tissue elastance (H) followed a hyperbolic distribution between a minimum and maximum. To test the model, we measured the input impedance of the respiratory system of ventilated normal and emphysematous C57BL/6 mice in closed chest condition at four levels of positive end-expiratory pressures. Mild emphysema was developed by nebulized porcine pancreatic elastase (PPE) (30 IU/day x 6 days). Respiratory mechanics were studied 3 wk following the initial treatment. The model significantly improved the fitting error compared with a single-compartment model. The PPE treatment was associated with an increase in mean alveolar diameter and a decrease in minimum, maximum, and mean H. The coefficient of variation of H was significantly larger in emphysema (40%) than that in control (32%). These results indicate that PPE treatment resulted in increased time-constant inequalities associated with a wider distribution of H. The heterogeneity of alveolar size (diameters and area) was also larger in emphysema, suggesting that the model-based tissue elastance heterogeneity may reflect the underlying heterogeneity of the alveolar structure.

title: Mitogen-Activated Protein Kinase Phosphatase-1 Negatively Regulates the Expression of Interleukin-6, Interleukin-8, and Cyclooxygenase-2 in A549 Human Lung Epithelial Cells
abstract: Mitogen-activated protein kinase phosphatase (MKP)-1 is a protein phosphatase that regulates the activity of p38 mitogen-activated protein (MAP) kinase and c-Jun NH2-terminal kinase (JNK) and, to lesser extent, p42/44 extracellular signal-regulated kinase. Studies with MKP-1(−/−) mice show that MKP-1 is a regulating factor suppressing excessive cytokine production and inflammatory response. The data on the role of MKP-1 in the regulation of inflammatory gene expression in human cells are much more limited. In the present study, we investigated the effect of MKP-1 on the expression of interleukin (IL)-6, IL-8 and cyclooxygenase (COX)-2 in response to stimulation with cytokines (tumor necrosis factor, IL-1β, and interferon-γ; 10 ng/ml each) in A549 human lung epithelial cells. Cytokines enhanced p38 and JNK phosphorylation and MKP-1 expression. p38 MAP kinase inhibitors 4-[4-(4-fluorophenyl)-5-(4-pyridinyl)-1H-imidazol-2-yl] phenol (SB202190) and 1-(5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl)-3(4-(2-morpholin-4-yl-ethoxy)naphthalen-1-yl)urea (BIRB 796) inhibited cytokine-induced phosphorylation of p38 substrate MAP kinase-activated protein kinase 2 and expression of IL-6, IL-8, and COX-2. An aminopyridine-based JNK inhibitor, N-(4-amino-5-cyano-6-ethoxypyridin-2-yl)-2-(2,5-dimethoxyphenyl)acetamide (JNK inhibitor VIII), inhibited phosphorylation of a JNK substrate c-Jun but did not have any effect on IL-6, IL-8, or COX-2 expression. Down-regulation of MKP-1 with small interfering RNA enhanced p38 and JNK phosphorylation and increased IL-6, IL-8, and COX-2 expression in A549 cells. In conclusion, cytokine-induced MKP-1 expression was found to negatively regulate p38 phosphorylation and the expression of IL-6, IL-8, and COX-2 in human pulmonary epithelial cells. Our results suggest that MKP-1 is an important negative regulator of inflammatory gene expression in human pulmonary epithelial cells, and compounds that enhance MKP-1 may have anti-inflammatory effects and control inflammatory response in the human lung.

title: A cell-active inhibitor of mitogen-activated protein kinase phosphatases restores paclitaxel-induced apoptosis in dexamethasone-protected cancer cells
abstract: Mitogen-activated protein kinase phosphatase (MKP)-1 is a dual-specificity phosphatase that negatively regulates the activity of mitogen-activated kinases and that is overexpressed in human tumors. Contemporary studies suggest that induction of MKP-1 during chemotherapy may limit the efficacy of clinically used antineoplastic agents. Thus, MKP-1 is a rational target to enhance anticancer drug activity, but suitable small-molecule inhibitors of MKP-1 are currently unavailable. Here, we have used a high-content, multiparameter fluorescence-based chemical complementation assay for MKP activity in intact mammalian cells to evaluate the cellular MKP-1 and MKP-3 inhibitory activities of four previously described, quinone-based, dual-specificity phosphatase inhibitors, that is, NSC 672121, NSC 95397, DA-3003-1 (NSC 663284), and JUN-1111. All compounds induced formation of reactive oxygen species in mammalian cells, but only one (NSC 95397) inhibited cellular MKP-1 and MKP-3 with an IC50 of 13 μmol/L. Chemical induction of MKP-1 by dexamethasone protected cells from paclitaxel-induced apoptosis but had no effect on NSC 95397. NSC 95397 phenocopied the effects of MKP-1 small inhibitory RNA by reversing the cytoprotective effects of dexamethasone in paclitaxel-treated cells. Isobologram analysis revealed synergism between paclitaxel and NSC 95397 only in the presence of dexamethasone. The data show the power of a well-defined cellular assay for identifying cell-active inhibitors of MKPs and support the hypothesis that small-molecule inhibitors of MKP-1 may be useful as antineoplastic agents under conditions of high MKP-1 expression.

title: TLR4 signaling in effector CD4+ T cells regulates TCR activation and experimental colitis in mice.
abstract: TLRs sense various microbial products. Their function has been best characterized in DCs and macrophages, where they act as important mediators of innate immunity. TLR4 is also expressed on CD4+ T cells, but its physiological function on these cells remains unknown. Here, we have shown that TLR4 triggering on CD4+ T cells affects their phenotype and their ability to provoke intestinal inflammation. In a model of spontaneous colitis, Il10-/-Tlr4-/- mice displayed accelerated development of disease, with signs of overt colitis as early as 8 weeks of age, when compared with Il10-/- and Il10-/-Tlr9-/- mice, which did not develop colitis by 8 months. Similar results were obtained in a second model of colitis in which transfer of naive Il10-/-Tlr4-/- CD4+ T cells into Rag1-/- recipients sufficient for both IL-10 and TLR4 induced more aggressive colitis than the transfer of naive Il10-/- CD4+ T cells. Mechanistically, LPS stimulation of TLR4-bearing CD4+ T cells inhibited ERK1/2 activation upon subsequent TCR stimulation via the induction of MAPK phosphatase 3 (MKP-3). Our data therefore reveal a tonic inhibitory role for TLR4 signaling on subsequent TCR-dependent CD4+ T cell responses.

title: Granulocyte-Macrophage Colony-Stimulating Factor Is a Chemoattractant Cytokine for Human Neutrophils: Involvement of the Ribosomal p70 S6 Kinase Signaling Pathway 1
abstract: GM-CSF stimulates proliferation of myeloid precursors in bone marrow and primes mature leukocytes for enhanced functionality. We demonstrate that GM-CSF is a powerful chemotactic and chemokinetic agent for human neutrophils. GM-CSF-induced chemotaxis is time dependent and is specifically neutralized with Abs directed to either the ligand itself or its receptor. Maximal chemotactic response was achieved at ∼7 nM GM-CSF, and the EC50 was ∼0.9 nM. Both concentrations are similar to the effective concentrations of IL-8 and less than the effective concentrations of other neutrophil chemoattractants such as neutrophil-activating peptide-78, granulocyte chemotactic protein-2, leukotriene B4, and FMLP. GM-CSF also acts as a chemoattractant for native cells bearing the GM-CSF receptor, such as monocytes, as well as for GM-CSF receptor-bearing myeloid cell lines, HL60 (promyelomonocyte leukemic cell line) and MPD (myeloproliferative disorder cell line), following differentiation induction. GM-CSF induced a rapid, transient increase in F-actin polymerization and the formation of focal contact rings in neutrophils, which are prerequisites for cell migration. The mechanism of GM-CSF-induced chemotaxis appears to involve the cell signaling molecule, ribosomal p70 S6 kinase (p70S6K). Both p70S6K enzymatic activity and T421/S424 and T389 phosphorylation are markedly increased with GM-CSF. In addition, the p70S6K inhibitor hamartin transduced into cells as active protein, interfered with GM-CSF-dependent migration, and attenuated p70S6K phosphorylation. These data indicate that GM-CSF exhibits chemotactic functionality and suggest new avenues for the investigation of the molecular basis of chemotaxis as it relates to inflammation and tissue injury.

title: Thioredoxin 1 delivery as new therapeutics.
abstract: Thioredoxin 1 (Trx 1) is a redox-active small protein ubiquitously present in human body. It is one of the defensive proteins induced in response to various stress conditions. In addition to its anti-oxidative effect by dithiol-disulfide exchange in its active site, Trx 1 has anti-apoptotic and anti-inflammatory effects. Trx 1 overexpression has been shown to be effective in a wide variety of animal models for oxidative and inflammatory disorders. An administration of recombinant Trx 1 protein is also effective in animal models especially for severe acute lung diseases where Trx 1 is likely to act with its anti-inflammatory properties. Trx 1 in circulation shows anti-chemotactic effects for neutrophils and inhibitory effects against macrophage migration inhibitory factor (MIF). Neovascularization is also suppressed by Trx 1 via inhibition of the complement activation. Here we discuss precise mechanisms of Trx 1 and potential therapeutic approach of this molecule.

title: Increased Expression of SVCT2 in a New Mouse Model Raises Ascorbic Acid in Tissues and Protects against Paraquat-Induced Oxidative Damage in Lung
abstract: A new transgenic mouse model for global increases in the Sodium Dependent Vitamin C transporter 2 (SVCT2) has been generated. The SVCT2-Tg mouse shows increased SVCT2 mRNA levels in all organs tested and correspondingly increased ascorbic acid (ASC) levels in all organs except liver. The extent of the increase in transporter mRNA expression differed among mice and among organs. The increased ASC levels did not have any adverse effects on behavior in the SVCT2-Tg mice, which did not differ from wild-type mice on tests of locomotor activity, anxiety, sensorimotor or cognitive ability. High levels of SVCT2 and ASC were found in the kidneys of SVCT2-Tg mice and urinary albumin excretion was lower in these mice than in wild-types. No gross pathological changes were noted in kidneys from SVCT2-Tg mice. SVCT2 immunoreactivity was detected in both SVCT2 and wild-type mice, and a stronger signal was seen in tubules than in glomeruli. Six treatments with Paraquat (3x10 and 3x15 mg/kg i.p.) were used to induce oxidative stress in mice. SVCT2-Tg mice showed a clear attenuation of Paraquat-induced oxidative stress in lung, as measured by F2-isoprostanes. Paraquat also decreased SVCT2 mRNA signal in liver, lung and kidney in SVCT2-Tg mice.

title: Neutralizing granulocyte/macrophage colony-stimulating factor inhibits cigarette smoke-induced lung inflammation.
abstract: Cigarette smoke is the major cause of chronic obstructive pulmonary disease (COPD), and there is currently no satisfactory therapy to treat people with COPD. We have previously shown that granulocyte/macrophage colony-stimulating factor (GM-CSF) regulates lung innate immunity to LPS through Akt/Erk activation of nuclear factor-kappaB and activator protein (AP)-1.



The aim of this study was to determine whether neutralization of GM-CSF can inhibit cigarette smoke-induced lung inflammation in vivo.



Male BALB/c mice were exposed to cigarette smoke generated from 9 cigarettes per day for 4 days. Mice were treated intranasally with 100 microg 22E9 (anti-GM-CSF mAb) and isotype control antibody on Days 2 and 4, 1 hour before cigarette smoke or sham exposure. On the fifth day mice were killed, and the lungs were lavaged with PBS and then harvested for genomic and proteomic analysis.



Cigarette smoke-exposed mice treated with anti-GM-CSF mAb had significantly less BALF macrophages and neutrophils, whole lung TNF-alpha, macrophage inflammatory protein (MIP)-2, and matrix metalloproteinase (MMP)-12 mRNA expression and lost less weight compared with smoke-exposed mice treated with isotype control. In contrast, smoke-induced increases in MMP-9 and net gelatinase activity were unaffected by treatment with anti-GM-CSF. In addition, neutralization of GM-CSF did not affect the phagocytic function of alveolar macrophages.



GM-CSF is a key mediator in smoke-induced airways inflammation, and its neutralization may have therapeutic implications in diseases such as COPD.

title: Essential Role of MAPK Phosphatase-1 in the Negative Control of Innate Immune Responses
abstract: TLR-induced innate immunity and inflammation are mediated by signaling cascades leading to activation of the MAPK family of Ser/Thr protein kinases, including p38 MAPK, which controls cytokine release during innate and adoptive immune responses. Failure to terminate such inflammatory reactions may lead to detrimental systemic effects, including septic shock and autoimmunity. In this study, we provide genetic evidence of a critical and nonredundant role of MAPK phosphatase (MKP)-1 in the negative control of MAPK-regulated inflammatory reactions in vivo. MKP-1−/− mice are hyperresponsive to low-dose LPS-induced toxicity and exhibit significantly increased serum TNF-α, IL-6, IL-12, MCP-1, IFN-γ, and IL-10 levels after systemic administration of LPS. Furthermore, absence of MKP-1 increases systemic levels of proinflammatory cytokines and exacerbates disease development in a mouse model of rheumatoid arthritis. When activated through TLR2, TLR3, TLR4, TLR5, and TLR9, bone marrow-derived MKP-1−/− macrophages exhibit increased cytokine production and elevated expression of the differentiation markers B7.2 (CD86) and CD40. MKP-1-deficient macrophages also show enhanced constitutive and TLR-induced activation of p38 MAPK. Based on these findings, we propose that MKP-1 is an essential component of the intracellular homeostasis that controls the threshold and magnitude of p38 MAPK activation in macrophages, and inflammatory conditions accentuate the significance of this regulatory function.

title: Susceptibility to exacerbation in chronic obstructive pulmonary disease.
abstract: Although we know that exacerbations are key events in chronic obstructive pulmonary disease (COPD), our understanding of their frequency, determinants, and effects is incomplete. In a large observational cohort, we tested the hypothesis that there is a frequent-exacerbation phenotype of COPD that is independent of disease severity.



We analyzed the frequency and associations of exacerbation in 2138 patients enrolled in the Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints (ECLIPSE) study. Exacerbations were defined as events that led a care provider to prescribe antibiotics or corticosteroids (or both) or that led to hospitalization (severe exacerbations). Exacerbation frequency was observed over a period of 3 years.



Exacerbations became more frequent (and more severe) as the severity of COPD increased; exacerbation rates in the first year of follow-up were 0.85 per person for patients with stage 2 COPD (with stage defined in accordance with Global Initiative for Chronic Obstructive Lung Disease [GOLD] stages), 1.34 for patients with stage 3, and 2.00 for patients with stage 4. Overall, 22% of patients with stage 2 disease, 33% with stage 3, and 47% with stage 4 had frequent exacerbations (two or more in the first year of follow-up). The single best predictor of exacerbations, across all GOLD stages, was a history of exacerbations. The frequent-exacerbation phenotype appeared to be relatively stable over a period of 3 years and could be predicted on the basis of the patient's recall of previous treated events. In addition to its association with more severe disease and prior exacerbations, the phenotype was independently associated with a history of gastroesophageal reflux or heartburn, poorer quality of life, and elevated white-cell count.



Although exacerbations become more frequent and more severe as COPD progresses, the rate at which they occur appears to reflect an independent susceptibility phenotype. This has implications for the targeting of exacerbation-prevention strategies across the spectrum of disease severity.