Han Pin Kuo

Neutrophil elastase induces IL-8 synthesis by lung epithelial cells via the mitogen-activated protein kinase pathway.

The sequestration of neutrophils in the lung and the release of proinflammatory mediators, including neutrophil elastase, are responsible for sepsis-induced microvascular permeability and alveolar epithelial cell damage. To assess the underlying mechanism, human neutrophil elastase (0.01–0.5 µg/ml) was added to cultured A549 epithelial cells in the presence or absence of inhibitors. IL-8 was analyzed by ELISA or by RT-PCR to measure the IL-8 synthesis capacity. Mitogen-activated protein kinase (MAPK) activity was detected by Western blot analysis. Neutrophil elastase dose-dependently increased IL-8 release from cultured A549 epithelial cells. Pretreatment with a specific elastase inhibitor, elastase inhibitor II (at 0.5, 5, and 50 µg/ml), dose-dependently inhibited neutrophil elastase-induced IL-8 release. The activities of MAPK, p38, and extracellular signal-regulated kinase (ERK) were upregulated by neutrophil elastase. Nuclear transcriptional factor-kappa B (NF-ĸB) and activator protein 1 (AP-1) were also activated. These responses were significantly inhibited by elastase inhibitor II. A specific inhibitor of p38 MAPK (SB203580) and an NF-ĸB inhibitor (pyrrolidine dithiocarbamate), but not an ERK inhibitor (PD 98059), significantly inhibited neutrophil elastase-induced IL-8 release and mRNA expression. The specific tyrosine kinase inhibitor, genistein, and the protein kinase C (PKC) inhibitor, Ro 31-8220, also inhibited IL-8 release and mRNA expression as well as p38 and NF-ĸB activation. There was no significant effect by the protein kinase A inhibitor, H-89, on neutrophil elastase-induced IL-8 synthesis or p38 MAPK activation. Our results indicate that neutrophil elastase activates p38 MAPK which upregulates NF-ĸB and AP-1 activities, thus inducing IL-8 mRNA expression and protein synthesis. Tyrosine kinase and PKC are implicated in neutrophil elastase activation of the MAPK pathway.

Tumor-associated macrophages correlate with response to epidermal growth factor receptor-tyrosine kinase inhibitors in advanced non-small cell lung cancer.

Our study investigated whether tumor‐associated macrophages (TAMs) in advanced non‐small cell lung cancer (NSCLC) are related to treatment response to epidermal growth factor receptor‐tyrosine kinase inhibitors (EGFR‐TKIs) and may be a predictor of survival. Of 206 advanced NSCLC patients treated (first‐line) with an EGFR‐TKI at the study hospital from 2006 to 2009, 107 with adequate specimens for assessing CD68 immunohistochemistry as a marker of TAMs were assessed. After EGFR‐TKI treatment, response was observed in 55 (51%) patients, and the median follow‐up period was 13.5 months. Most TAMs were located in the tumor stroma (>95%) and positively costained with the M2 marker CD163. TAM counts were significantly higher in patients with progressive disease than in those without (p < 0.0001), a trend that remained in patients with known EGFR mutation status (n = 59) and those with wild‐type EGFR (n = 20). High TAM counts, among other factors (e.g., wild‐type EGFR), were significantly related to poor progression‐free survival (PFS) and overall survival (OS) (all p < 0.0001 for TAMs). Multivariate Cox analyses showed that high TAM counts and EGFR mutations were both independent factors associated with PFS [odds ratio (OR), 8.0; 95% confidence interval (CI), 2.87–22.4; p = 0.0001 and OR, 0.03; 95% CI, 0.003–0.31; p = 0.003, respectively] and OS (OR, 2.641; 95% CI, 1.08–6.5; p = 0.03 and OR, 0.14; 95% CI, 0.03–0.56; p = 0.006, respectively). TAMs are related to treatment response irrespective of EGFR mutation and can independently predict survival in advanced NSCLC treated with an EGFR‐TKI.

Increased activation of fibrocytes in patients with chronic obstructive asthma through an epidermal growth factor receptor–dependent pathway

BACKGROUNDnFibrocytes are circulating progenitor cells that are increased in asthmatic patients with chronic obstructive asthma (COA) and rapid decrease in lung function. Fibrocytes from patients with COA have a greater capacity for proliferation and differentiation.nnnOBJECTIVEnWe investigated whether epidermal growth factor receptor (EGFR) activation mediated the proliferation of fibrocytes in patients with COA and whether oxidative stress was involved in this activation.nnnMETHODSnCirculating fibrocytes from nonadherent non-T-cell mononuclear cell fractions from healthy subjects, asthmatic patients with normal pulmonary function, and patients with COA were determined by using flow cytometric coexpression of collagen I, CD45, and CD34 or EGFR or a disintegrin and metalloprotease domain 17 and placed in culture.nnnRESULTSnExpression of EGFR was increased in fibrocytes from patients with COA compared with that seen in patients with NPF. AG1478 and gefitinib, inhibitors of EGFR tyrosine kinase, reduced fibrocyte proliferation and myofibroblast transformation. Increased expression of EGFR and fibrocyte proliferation and transformation were induced by hydrogen peroxide, and these effects were inhibited by N-acetylcysteine.nnnCONCLUSIONSnEnhanced fibrocyte proliferation and transformation found in patients with COA might be mediated through an oxidant-sensitive EGFR-dependent pathway.

Persistence of lung inflammation and lung cytokines with high-resolution CT abnormalities during recovery from SARS

BackgroundDuring the acute phase of severe acute respiratory syndrome (SARS), mononuclear cells infiltration, alveolar cell desquamation and hyaline membrane formation have been described, together with dysregulation of plasma cytokine levels. Persistent high-resolution computed tomography (HRCT) abnormalities occur in SARS patients up to 40 days after recovery.MethodsTo determine further the time course of recovery of lung inflammation, we investigated the HRCT and inflammatory profiles, and coronavirus persistence in bronchoalveolar lavage fluid (BALF) of 12 patients at recovery at 60 and 90 days.ResultsAt 60 days, compared to normal controls, SARS patients had increased cellularity of BALF with increased alveolar macrophages (AM) and CD8 cells. HRCT scores were increased and correlated with T-cell numbers and their subpopulations, and inversely with CD4/CD8 ratio. TNF-α, IL-6, IL-8, RANTES and MCP-1 levels were increased. Viral particles in AM were detected by electron microscopy in 7 of 12 SARS patients with high HRCT score. On day 90, HRCT scores improved significantly in 10 of 12 patients, with normalization of BALF cell counts in 6 of 12 patients with repeat bronchoscopy. Pulse steroid therapy and prolonged fever were two independent factors associated with delayed resolution of pneumonitis, in this non-randomized, retrospective analysis.ConclusionResolution of pneumonitis is delayed in some patients during SARS recovery and may be associated with delayed clearance of coronavirus, Complete resolution may occur by 90 days or later.

Fibrocyte trafficking in patients with chronic obstructive asthma and during an acute asthma exacerbation.

BACKGROUNDnFibrocytes express several chemokine receptors (CCR7 and CXCR4) that regulate their recruitment and trafficking into tissue-damage sites in response to specific chemokine gradients (CCL19 and CXCL12).nnnOBJECTIVEnWe investigated whether these chemoattractants and S100A9, through the receptor for advanced glycation end-products (RAGE; ie, its receptor), are involved in fibrocyte trafficking in patients with chronic obstructive asthma (COA) and during an acute exacerbation (AE) in patients without airflow obstruction (Asthma AE group).nnnMETHODSnWe collected peripheral blood from 14 asthmatic patients with normal pulmonary function, 14 patients with COA, 11 patients in the Asthma AE group, and 14 healthy subjects. Isolated circulating fibrocytes were used for migration assay. Expression of CCR7, CXCR4, S100A9, and RAGE in fibrocytes was measured by using flow cytometry. CCL19 and CXCL12 expression in bronchial tissues was determined by using immunohistochemistry and RT-PCR.nnnRESULTSnThere were higher numbers of circulating fibrocytes in patients in the Asthma AE group and patients with COA. The expression of CXCL12 in bronchial tissues and CXCR4 in circulating fibrocytes was higher in the Asthma AE group and, to a lesser extent, in patients with COA. The expression of CCL19 in bronchial tissues and CCR7 in fibrocytes was higher in patients with COA. CXCL12/CXCR4 and CCL19/CCR7 enhanced fibrocyte transmigration in the Asthma AE group and in patients with COA, respectively. The upregulated expression of S100A9 and RAGE in fibrocytes of patients in the Asthma AE group and those with COA contributes to the enhanced basal migratory motility of fibrocytes.nnnCONCLUSIONnThe CXCR4/CXCL12 axis contributes to chemotaxis of fibrocytes in patients in the Asthma AE group, whereas the CCR7/CCL19 axis plays an important role in patients with COA. S100A9 enhances the basal migratory motility of fibrocytes from patients in the Asthma AE group and patients with COA.

Concomitant Active Tuberculosis Prolongs Survival in Non-Small Cell Lung Cancer: A Study in a Tuberculosis-Endemic Country

Background Adjuvant tumor cell vaccine with chemotherapy against non-small cell lung cancer (NSCLC) shows limited clinical response. Whether it provokes effective cellular immunity in tumor microenvironment is questionable. Concomitant active tuberculosis in NSCLC (TBLC) resembles locoregional immunotherapy of tumor cell vaccine; thus, maximally enriches effective anti-tumor immunity. This study compares the survival and immunological cell profile in TBLC over NSCLC alone. Methods Retrospective review of NSCLC patients within 1-year-period of 2007 and follow-up till 2010. Results A total 276 NSCLC patients were included. The median survival of TBLC is longer than those of NSCLC alone (11.6 vs. 8.8 month, p<0.01). Active tuberculosis is an independent predictor of better survival with HR of 0.68 (95% CI, 0.48∼0.97). Squamous cell carcinoma (SCC) (55.8 vs. 31.7%, p<0.01) is a significant risk factor for NSCLC with active TB. The median survival of SCC with active tuberculosis is significantly longer than adenocarcinoma or undetermined NSCLC with TB (14.2 vs. 6.6 and 2.8 months, p<0.05). Active tuberculosis in SCC increases the expression of CD3 (46.4±24.8 vs. 24.0±16.0, p<0.05), CXCR3 (35.1±16.4 vs. 19.2±13.3, p<0.01) and IP-10 (63.5±21.9 vs. 35.5±21.0, p<0.01), while expression of FOXP3 is decreased (3.5±0.5 vs. 13.3±3.7 p<0.05, p<0.05). Survival of SCC with high expression of CD3 (12.1 vs. 3.6 month, p<0.05) and CXCR3 (12.1 vs. 4.4 month, p<0.05) is longer than that with low expression. Conclusions Active tuberculosis in NSCLC shows better survival outcome. The effective T lymphocyte infiltration in tumor possibly underlies the mechanism. Locoregional immunotherapy of tumor cell vaccine may deserve further researches.

Mobile-phone-based home exercise training program decreases systemic inflammation in COPD: a pilot study

BackgroundModerate-intensity exercise training improves skeletal muscle aerobic capacity and increased oxidative enzyme activity, as well as exercise tolerance in COPD patients.MethodsTo investigate whether the home-based exercise training program can reduce inflammatory biomarkers in patients with COPD, twelve patients using mobile phone assistance and 14 with free walk were assessed by incremental shuttle walk test (ISWT), spirometry, strength of limb muscles, and serum C-reactive protein (CRP) and inflammatory cytokines.ResultsPatients in the mobile phone group improved their ISWT walking distance, with decrease in serum CRP after 2 months, and sustained at 6 months. Patients in the control group had no improvement. Serum IL-8 in the mobile phone group was significantly reduced at 2, 3 and 6 months after doing home exercise training compared to baseline. IL-6 and TNF-α were significantly elevated at 3 and 6 months in control group, while there were no changes in mobile phone group. The strength of limb muscles was significantly greater compared to baseline at 3 and 6 months in the mobile phone group.ConclusionsA mobile-phone-based system can provide an efficient home endurance exercise training program with improved exercise capacity, strength of limb muscles and a decrease in serum CRP and IL-8 in COPD patients. Decreased systemic inflammation may contribute to these clinical benefits. (Clinical trial registration No.: NCT01631019)

Endogenous nitric oxide downregulates the Bcl-2 expression of eosinophils through mitogen-activated protein kinase in bronchial asthma

BACKGROUNDnEosinophil apoptosis might play a crucial role in the maintenance of persistent airway inflammation in asthma. Nitric oxide (NO) synthase activity is upregulated in eosinophils of asthmatic patients. Mitogen-activated protein kinase (MAPK) is implicated in regulating eosinophil apoptosis by modulating Bcl-2 expression. NO-induced cell apoptosis is associated with an inhibition of Bcl-2 expression.nnnOBJECTIVEnWe sought to study whether NO might induce eosinophil apoptosis through extracellular signal-regulated protein kinase (ERK) or p38 MAPK pathways and Bcl-2 expression.nnnMETHODSnEosinophils were freshly isolated from peripheral blood of 16 asthmatic patients and 12 healthy subjects and then cultured with or without the NO synthase inhibitor N-nitro-l-arginine methyl ester (L-NAME) at 1 and 10 mmol/L for 16 hours. The expression of Bcl-2 and induced NO synthase on eosinophils was analyzed by means of flow cytometry. Apoptosis was assessed by means of propidium iodide and DNA ladder. The activity of ERK and p38 MAPK was determined by means of Western blotting.nnnRESULTSnThe induced NO synthase immunoreactivities and the spontaneous release of nitrite from the eosinophils of asthmatic patients were higher compared with those of healthy subjects. Eosinophils of asthmatic patients were found to express more highly constitutive Bcl-2 than those of healthy subjects. After incubation for 16 hours, the expression of Bcl-2 on eosinophils from patients with asthma was significantly enhanced by L-NAME. The percentage of apoptosis was decreased by the addition of 1 mmol/L L-NAME in patients with asthma. The activity of p38 MAPK and ERK in eosinophils from patients with asthma was enhanced in the presence of L-NAME. An inhibition of MAPK reduced the Bcl-2 expression and increased eosinophil apoptosis in patients with asthma.nnnCONCLUSIONnWe concluded that inhibition of endogenous NO might increase the expression of Bcl-2 in eosinophils from patients with asthma through the signaling pathway of ERK or p38 MAPK, which in turn decrease the apoptosis.

NF-κB repressing factor downregulates basal expression and mycobacterium tuberculosis induced IP-10 and IL-8 synthesis via interference with NF-κB in monocytes

BackgroundOur previous study showed NF-κB repressing factor (NKRF) downregulates IP-10 and IL-8 synthesis in the peripheral blood mononuclear cells and alveolar macrophages of TB patients with high bacterial loads. However, the mechanism underlying the repressive effect of NKRF is not fully understood.ResultsThe levels of IP-10, IL-8 and NKRF were significantly up-regulated in THP-1 cells treated with heated mycobacterium tuberculosis (H. TB). NKRF inhibited NF-κB-mediated IP-10 and IL-8 synthesis and release induced by H. TB. The repressive effect of NKRF is mediated via interference with NF-κB (p65) binding and RNA polymerase II recruitment to promoter sites of IP-10 and IL-8.ConclusionsWe have elucidated that direct contact with MTb induces IP-10, IL-8 and a concomitant increase in NKRF in THP-1 cells. The up-regulated NKRF serves as an endogenous repressor for IP-10 and IL-8 synthesis to hinder host from robust response to MTb infection.

Effect of theophylline and specific phosphodiesterase IV inhibition on proliferation and apoptosis of progenitor cells in bronchial asthma

Theophylline possesses anti‐inflammatory activities in asthma. We examined whether theophylline and agents that modulate cyclic AMP can determine the survival and proliferation of progenitor cells. Progenitor cells from the blood of normal and asthmatic subjects were cultured for 14 days in methylcellulose with GM‐CSF, stem cell factor, IL‐3 and IL‐5. Apoptosis was measured by flow cytometry of propidium‐iodide‐stained cells. A greater number of colonies with a higher proportion of cells of eosinophil lineage from asthmatics compared to normal subjects were grown. Theophylline (at 5 and 20 μg ml−1) significantly inhibited colony formation and increased apoptotic cells in asthmatics compared to control. Salbutamol (0.1, 1, 10 μM), dibutyryl‐cAMP (0.1, 1 mM) and rolipram (0.1, 1 mM), a phosphodiesterase IV inhibitor, also dose‐dependently decreased colony numbers and increased apoptosis of progenitor cells from asthmatics. There was no significant effect of theophylline, db‐cAMP, salbutamol or rolipram on colony formation or the survival of progenitor cells from normal subjects. AMP did not affect the colony formation and apoptosis. Expression of Bcl‐2 protein on progenitor cells of asthma was downregulated by theophylline, salbutamol, db‐cAMP and rolipram. Theophylline and rolipram decreased colony formation committed to the eosinophil lineage, together with an increase in apoptosis through an inhibition of Bcl‐2 expression effects that may occur through cAMP. The anti‐inflammatory properties of theophylline include an inhibition of circulating progenitor cells.