Nasal IL-17F is related to bronchial IL-17F/neutrophilia and exacerbations in stable atopic severe asthma.

Severe asthma (SA) is associated with neutrophil recruitment and T helper (TH )17 chemokine overexpression in bronchial biopsies. We aimed to evaluate IL-17A and IL-17F expression in nasal/bronchial lamina propria of atopic mild-to-severe asthmatics and controls in relation to neutrophilia and asthma exacerbations. Cryostat sections of nasal/bronchial biopsies obtained from 14 SA and 14 mild asthma (MA) stable atopic patients with rhinitis, and seven healthy controls were analyzed by immunohistochemistry for neutrophils, IL-17A and IL-17F expression. Atopic SA showed an increase in asthma exacerbations number, IL-17F and IL-17A expression in nasal/bronchial lamina propria compared to MA and controls, and a higher expression of bronchial neutrophils in SA compared to MA and controls. In all asthmatics, significant relationships were found between bronchial IL-17F and neutrophils/FEV1 , nasal IL-17F and bronchial neutrophil/IL-17 markers and between the latter and exacerbations, suggesting that nasal IL-17F might be informative on bronchial IL17-driven neutrophilia in atopic SA.

The role of transforming growth factor-ß1 in airway inflammation of childhood asthma.

TGF-beta-targeting structural and inflammatory cells has been implicated in the mechanisms leading to the inflammatory and restructuring processes in asthma, suggesting an impact of TGF-beta1 signaling on the development and persistency of this disease. We investigated the potential early involvement of TGF-beta1 activity in the immunological and molecular mechanisms underlying progression of inflammation in childhood asthma. We evaluated the levels of TGF-beta1 in induced sputum supernatants (ISSs) and the expression of small mother cell against decapentaplegic (Smad) 2 and Smad7 proteins in induced sputum cells (ISCs) from children with intermittent asthma (IA), moderate asthma (MA) and control subjects (C). Furthermore, we investigated the regulatory role of TGF-beta1 activity on eosinophil and neutrophil adhesion to epithelial cells using adhesion assay, and on the granulocyte expression of adhesion molecule CD11b/CD18 Macrophage-1 antigen (MAC-1), by flow cytometry. We found that the levels of TGF-beta1 are increased in ISSs of IA and MA in comparison to C, concomitantly to the activation of intracellular signaling TGFbeta/Smads pathway in ISCs. In MA, TGF-beta1 levels correlated with the number of sputum eosinophils and neutrophils. Furthermore, we showed the ability of sputum TGF-beta1 to promote eosinophil and neutrophil adhesion to epithelial cells, and to increase the expression of MAC-1 on the granulocyte surface. This study shows the activation of TGFbeta/Smad signaling pathway in the airways of children with IA and, despite the regular ICS treatment, in children with MA, and provides evidence for the contribution of TGF-beta1 in the regulation of granulocyte activation and trafficking.

Plasma leptin and vascular endothelial growth factor (VEGF) in normal subjects at high altitude (5050 m).

CONTEXT: High altitude (HA) is a model of severe hypoxia exposure in humans. We hypothesized that nocturnal hypoxemia or acute maximal exercise at HA might affect plasma leptin and VEGF levels. OBJECTIVES: Plasma leptin, VEGF and other metabolic variables were studied after nocturnal pulse oximetry and after maximal exercise in healthy lowlanders on the 3rd-4th day of stay in Lobuche (5050 m, HA) and after return to sea level (SL). RESULTS: Leptin was similar at SL or HA in both pre- and post-exercise conditions. Pre-exercise VEGF at HA was lower, and cortisol was higher, than at SL, suggesting that nocturnal intermittent hypoxia associated with periodic breathing at HA might affect these variables. CONCLUSIONS: Leptin levels appear unaffected at HA, whereas nocturnal hypoxic stress may affect plasma VEGF. Future HA studies should investigate the possible role of nocturnal intermittent hypoxemia on metabolism.

Biochemical interaction between effects of beclomethasone dipropionate and salbutamol or formoterol in sputum cells from mild to moderate asthmatics.

BACKGROUND: Several in vitro studies demonstrate that corticosteroids and long-acting beta(2) agonists may have a complementary and synergistic mode of action on the inflammatory processes in asthma. METHODS: Sputum was induced in 20 mild to moderate asthmatic patients and the induced sputum cells (ISC) were cultured with beclomethasone dipropionate (BDP) 10(-7) M, salbutamol 10(-8) M and formoterol 10(-8) M either alone or in combination, BDP plus salbutamol and BDP plus formoterol, for 24 h. We measured the levels of growth macrophages-colony stimulating factor (GM-CSF), released on activation normal T cells expressed and activated (RANTES) and interleukin-8 (IL-8), in the supernatant of stimulated cells by ELISA. Furthermore, we assessed nuclear translocation of glucocorticoid receptor (GR) and the expression of beta(2) receptor in ISC by immunofluorescence and RT-PCR, respectively. RESULTS: The release of GM-CSF, RANTES and IL-8 in ISC was significantly reduced by BDP plus salbutamol or formoterol as compared with either drug alone (P < 0.0001). beta(2) receptor expression was increased after 30 min of incubation with BDP and continued to increase over a time period of 4 h (P < 0.0001). Furthermore after 30 min of incubation, nuclear translocation of GR was greater with BDP plus salbutamol or formoterol than with any of the drugs alone (P < 0.0001). CONCLUSION: The present ex vivo study demonstrates a complementary mode of action between BDP and salbutamol or formoterol leading to an enhanced anti-inflammatory activity.

LTB4 is present in exudative pleural effusions and contributes actively to neutrophil recruitment in the inflamed pleural space.

The pleural space is a virtual compartment between the lung and chest wall that becomes filled with fluid and inflammatory cells during a variety of respiratory diseases. Here, we study the potential role of the eicosanoid metabolite leukotriene B4 (LTB4) in disparate diseases leading to acute (pneumonia) or chronic (tuberculosis, cancer) inflammation of the pleural space. LTB4 concentrations were significantly higher in pleural fluid due to pneumonia, tuberculosis and cancer with respect to congestive heart failure and correlated with neutrophil elastase, which is used as an indication of state of activation of neutrophils in the pleural space. Moreover, pleural LTB4 was biologically active, as an anti-LTB4 antibody partially neutralized the chemotactic activity of parapneumonic, tuberculous and cancer effusions. Macrophages, neutrophils, lymphocytes, mesothelial cells and cancer cells all expressed mRNA for 5-lipoxygenase, the enzyme that initiates leukotriene synthesis leading to the production of LTB4, in exudative pleural effusions. Upon stimulation in transudative pleural effusions, pleural macrophages produced, in a time-dependent fashion, a significantly higher concentration of LTB4 than mesothelial cells. These studies demonstrate that different cell types are capable of producing LTB4 in the inflamed pleural space and that this mediator may play a crucial role in the recruitment of neutrophils into the pleural space.

Effect of cilomilast (Ariflo) on TNF-alpha, IL-8, and GM-CSF release by airway cells of patients with COPD.

BACKGROUND: Inflammation in chronic obstructive pulmonary disease (COPD) is characterised by increased neutrophilic infiltration of the airways. Cilomilast, a novel selective phosphodiesterase 4 inhibitor in clinical development for COPD treatment, exerts anti-inflammatory effects. The ability of cilomilast to inhibit the release of neutrophil chemoattractants such as tumour necrosis factor (TNF)-alpha, interleukin (IL)-8, and granulocyte-macrophage colony stimulating factor (GM-CSF) by bronchial epithelial cells and sputum cells isolated from 10 patients with COPD, 14 normal controls, and 10 smokers was investigated. METHODS: Bronchial epithelial cells obtained by bronchial brushing and sputum cells isolated from induced sputum samples were cultured for 24 hours in the presence or absence of cilomilast (1 micro M). After incubation the supernatants were harvested and the levels of mediators measured by ELISA. Chemotactic activity in supernatants was also measured using a Boyden chamber. RESULTS: TNF-alpha and IL-8 release by bronchial epithelial cells and sputum cells was higher in patients with COPD than in controls (p<0.0001) and smokers (p<0.0001). GM-CSF was only detectable in sputum cell supernatants and its level was higher in patients with COPD than in controls and smokers (p<0.0001, respectively). Cilomilast significantly reduced TNF-alpha release by bronchial epithelial cells and sputum cells (p=0.005) and GM-CSF release by sputum cells (p=0.003), whereas IL-8 release was not statistically inhibited. Supernatants of sputum cells and bronchial epithelial cells treated with cilomilast significantly decreased neutrophil chemotaxis (p<0.006 and p<0.008, respectively). CONCLUSIONS: Cilomilast inhibits the production of some neutrophil chemoattractants by airway cells. This drug may play a role in the resolution of neutrophilic inflammation associated with COPD and cigarette smoke.

Fluticasone induces apoptosis in peripheral T-lymphocytes: a comparison between asthmatic and normal subjects.

Apoptosis is an important mechanism allowing inflammation to be limited. Glucocorticoids are the most effective anti-inflammatory agents in asthma therapy and induce cell apoptosis. Since T-lymphocytes are critically involved in airway inflammation in asthma, the effects of fluticasone propionate (FP) on apoptosis in unstimulated and in interleukin (IL)-2 stimulated peripheral blood T-lymphocytes (PBTs) isolated from 14 normal and 19 mild-to-moderate asthmatic subjects were evaluated. Apoptosis was evaluated by: deoxyribonucleic acid (DNA) fragmentation electrophoresis, DNA content, annexin V binding, apoptosis related markers (Fas, B-cell lymphona leukaemia-2 (Bcl-2), Bax, and CD25), and by electron microscopy. FP induced apoptosis in unstimulated PBTs of normal and asthmatic subjects in a time-dependent fashion. In asthma, this effect was associated with a significant decrease of Bcl-2 expression, and with an increase of Bax/Bcl-2 ratio. In PBTs of asthmatics, FP also reduced Fas and CD25 expression. Moreover, in IL-2-stimulated PBTs from both asthmatics and normal subjects, FP was able to induce apoptosis and to reduce Bcl-2, Fas and CD25 expression, whereas negligible effects were detected on Bax expression. This study shows that the glucocorticosteroid, fluticasone, increases apoptosis and modulates expression of apoptosis-related markers in unstimulated and in interleukin-2 stimulated T-lymphocytes. This points towards a potential mechanism by which fluticasone exerts its anti-inflammatory effects.

15(S)-HETE modulates LTB(4) production and neutrophil chemotaxis in chronic bronchitis.

We evaluated the levels of 15(S)-hydroxyeicosatetraenoic acid [15(S)-HETE] and the expression of 15-lipoxygenase (15-LO) mRNA in induced sputum obtained from 10 control and 15 chronic bronchitis subjects. 15(S)-HETE was evaluated by reverse phase high-performance liquid chromatography separation followed by specific RIA. 15-LO mRNA expression was determined by primed in situ labeling. The levels of both soluble and cell-associated 15(S)-HETE resulted significantly higher in chronic bronchitis than in control subjects. The percentage of cells expressing 15-LO mRNA was significantly higher in chronic bronchitis than in control subjects (P < 0.01). Double staining for specific cell type markers and 15-LO mRNA showed macrophages and neutrophils positive for 15-LO, whereas similar staining of peripheral blood neutrophils did not show evidence for 15-LO expression, suggesting that expression of 15-LO in neutrophils takes place on migration into the airways. Because 15(S)-HETE inversely correlated with the percentage of neutrophils in sputum of chronic bronchitis subjects, we studied the effect of 15(S)-HETE on leukotriene B(4) (LTB(4)) production in vitro and evaluated the concentration of LTB(4) in induced sputum and the contribution of LTB(4) to the chemotactic activity of induced sputum samples ex vivo. The results obtained indicate that macrophages and neutrophils present within the airways of chronic bronchitis subjects express 15-LO mRNA; increased basal levels of 15(S)-HETE may contribute to modulate, through the inhibition of 5-lipoxygenase metabolites production, neutrophil infiltration and airway inflammation associated with chronic bronchitis.

Effects of gemcitabine on cell proliferation and apoptosis in non-small-cell lung cancer (NSCLC) cell lines.

We evaluated the antiproliferative and the proapoptotic ability of gemcitabine in three non-small-cell lung cancer (NSCLC) cell lines. NCI-H292 (mucoepidermoid carcinoma), NCI-CorL23 (large-cell carcinoma) and NCI-Colo699 (adenocarcinoma) cells were cultured with and without 0.5, 0.05 and 0.005 microM gemcitabine for 24, 48 and 72 h, respectively. Gemcitabine exerted a stronger and earlier antiproliferative and proapoptotic effect on H292 cells than on CorL23 or Colo699 cells. Fas receptor expression was increased in all three cell lines and was higher in Colo699 than in CorL23 cells. The incubation of NSCLC with anti-Fas agonistic monoclonal antibody (CH11) induced cell apoptosis in H292 cells, demonstrating that the Fas receptor was functionally active. Finally, gemcitabine and CH-11 exerted a synergistic effect on cell apoptosis in H292 cells. This study demonstrates that gemcitabine induces apoptosis in NSCLC and that this effect might be exerted by modulating functionally active Fas expression, and these effects of gemcitabine were stronger in H292 cells than in either CorL23 or Colo699 cells.

Interleukin-8 induces lymphocyte chemotaxis into the pleural space. Role of pleural macrophages.

The pleural space is a potential compartment between the lung and chest wall that becomes filled with fluid and inflammatory cells in a number of respiratory diseases. In an attempt to understand one aspect of the inflammatory process in the pleural space, we compared the responses in three different diseases (congestive heart failure [CHF], tuberculosis [TB], and cancer). Large concentrations of interleukin-8 (IL-8) were detected in cancer and TB effusions, but not in CHF. Surprisingly, the concentration of IL-8 correlated best with lymphocyte recruitment and not with neutrophil recruitment. Pleural fluid from cancer and TB patients was chemotactic for lymphocytes, and this activity was partly blocked by an anti-IL-8 antibody in cancer and completely blocked in TB. To determine whether there was the potential for a chemotactic gradient into the pleural space, pleural effusion cells were analyzed for the expression of IL-8. Cells in the effusions of cancer patients expressed IL-8, whereas IL-8 could not be detected from the cells of TB and CHF effusions. To explore the possible role of pleural macrophages in the regulation of IL-8, pleural effusion cells were treated with culture supernatants from stimulated pleural macrophages. Stimulated pleural macrophages were able to induce expression of messenger RNA (mRNA) for IL-8 and IL-8 protein production, and this activity was abrogated by blocking tumor necrosis factor-alpha. These findings suggest that soluble IL-8 is an important factor for the recruitment of lymphocytes into the pleural space, and that this cytokine is produced by both pleural structural and cancer cells after their activation by macrophage-derived, cytokine-mediated signals.

Evaluation of apoptosis of eosinophils, macrophages, and T lymphocytes in mucosal biopsy specimens of patients with asthma and chronic bronchitis.

BACKGROUND: Apoptosis regulates inflammatory cell survival, and its reduction contributes to the chronicity of an inflammatory process. Apoptosis is controlled by suppressing or inducing genes, such as bcl-2 and p53, respectively. OBJECTIVE: We sought to assess apoptosis of eosinophils, macrophages, and T lymphocytes in bronchial biopsy specimens from asthmatic subjects and to examine its regulation by evaluating the expression of B-cell lymphoma leukemia-2 (Bcl-2) and P53 proteins. We also sought to explore the relationships between cell apoptosis and GM-CSF, a cytokine able to increase eosinophil and macrophage survival. METHODS: Apoptosis in eosinophils, macrophages, and T lymphocytes was evaluated in bronchial biopsy specimens obtained from 30 asthmatic subjects, 26 subjects with chronic bronchitis, and 15 control subjects by combining the terminal deoxynucleotidyl transferase-mediated dNTP nick end-labeling technique and immunohistochemistry. The expression of P53, Bcl-2, and GM-CSF was studied through immunohistochemistry by using specific mAbs. RESULTS: The number of apoptotic eosinophils and macrophages was lower in subjects with asthma than in those with chronic bronchitis (P <.007 and P <.001, respectively) and inversely correlated with the clinical severity of asthma (P <.001 and P <.002, respectively). Few T lymphocytes were apoptotic in all groups studied. In asthma GM-CSF+ cells correlated with the number of nonapoptotic eosinophils and macrophages (P =.0001) and with the severity of the disease (P <.003). In asthma Bcl-2+ cells were higher than in control subjects and subjects with chronic bronchitis (P <.002 and P <.015, respectively), they outnumbered P53+ cells, and they correlated with the number of T lymphocytes (P <.001) and with the severity of the disease (P <.003). CONCLUSION: Airway inflammation in asthma is associated with an enhanced survival of different cell types caused by reduced apoptosis.

Interleukin-4 enhances 15-lipoxygenase activity and incorporation of 15(S)-HETE into cellular phospholipids in cultured pulmonary epithelial cells.

15(S)-Hydroxyeicosatetraenoic acid (15[S]-HETE) is a 15-lipoxygenase (15-LO) metabolite that may play an important role in different pulmonary diseases. 15-HETE is synthesized by different epithelial cells and may be subsequently incorporated into cellular phospholipids. We studied the role of interleukin-4 (IL-4) on 15-LO activity and on 15(S)-HETE incorporation into cellular phospholipids by WI-26 pulmonary epithelial cells. 15-LO activity was evaluated by measuring 15(S)-HETE production, through combined reverse-phase-high-pressure liquid chromatography (RP-HPLC) separation and specific radioimmunoassay (RIA), after incubation with arachidonic acid (AA). We also studied 15-LO messenger RNA (mRNA) expression, using primed in situ (PRINS) labeling. IL-4 (10 ng/ml) markedly increased the percentage of 15-LO mRNA-bearing cells as well as 15-LO activity after 24, 48, and 72 h, with a maximal response at 48 h. Uptake and incorporation into cellular phospholipid was studied with [3H]15(S)-HETE, which showed that IL-4 was able to increase significantly 15(S)-HETE incorporation into WI-26 cells, with a maximal effect observed at 72 h. Cellular-lipid-associated [3H]15(S)-HETE, evaluated with RP-HPLC after base-catalyzed hydrolysis, increased concomitantly with disappearance of the radiolabel from the supernatant. Class separation of cellular lipids with normal-phase HPLC (NP-HPLC) showed that IL-4 increased [3H]15(S)- HETE incorporation mainly in the phosphatidylinositol (PI) fraction. The ability of IL-4 to promote 15-LO activity and incorporation into cellular phospholipids of human lung epithelial cells may be important in airway inflammation and in modulation of the potential autocrine function of 15(S)-HETE.

Phenotypic and functional characterization of normal rat pleural macrophages in comparison with autologous peritoneal and alveolar macrophages.

Pleural mononuclear phagocytes (PleMP) were isolated from normal rats by pleural lavage and compared with autologous peritoneal (PerMP) and bronchoalveolar mononuclear phagocytes (BAMP) isolated by peritoneal and bronchoalveolar lavage, respectively. The phagocytic activity of PleMP, PerMP, and BAMP, evaluated by testing their ability to ingest latex beads, was lower for PleMP and PerMP than for BAMP. The phenotype of PleMP, PerMP, and BAMP was characterized by immunocytochemical staining with a panel of monoclonal antibodies (mAbs). As expected, PleMP, PerMP, and BAMP did not react with OX19, OX33, ED5, MOM/3F12/F2, and anticytokeratin mAbs, specific for T lymphocytes, B lymphocytes, dendritic cells, granulocytes, and epithelial/mesothelial cells, respectively. Moreover, PleMP and PerMP populations were highly enriched with OX6-, OX42-, ED7-, and ED8-positive MP, whereas BAMP population was enriched with ED1- and ED9-positive cells. To test the ability of PleMP, PerMP, and BAMP to function as accessory cells (AC), mitomycin C-treated MP were used as stimulatory cells in mixed leukocyte reaction experiments, using allogeneic T cells as responders. 3HdTR incorporation by T cells was assessed as an index of AC function. PleMP and PerMP were more potent AC than BAMP. Moreover, when cultured together with autologous pulmonary interstitial dendritic cells, PleMP and PerMP exerted a more potent ability to stimulate T-cell proliferation than did BAMP. To investigate the capacity of MP to function as bactericidal and fungicidal cells, we tested their ability to kill Escherichia coli and Cryptococcus neoformans, respectively. PleMP and PerMP were less potent bactericidal and fungicidal cells than BAMP. The results of this study demonstrate that PleMP isolated from normal rat pleural space are functionally and phenotypically different from BAMP but similar to PerMP, and suggest that these cells might play an important role in cell-mediated immune reactions in the pleural space.

Sputum metalloproteinase-9/tissue inhibitor of metalloproteinase-1 ratio correlates with airflow obstruction in asthma and chronic bronchitis.

Asthma and chronic bronchitis are inflammatory diseases with extracellular matrix (ECM) remodeling and collagen deposition. Collagen homeostasis is controlled by metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). We evaluated MMP and TIMP balance in induced sputum of 10 control, 31 untreated asthmatic, and 16 chronic bronchitic subjects. We first performed zymographic analysis to identify the profile of MMPs. Zymography revealed a similar MMPs profile in all populations studied and that MMP-9 was the major enzyme released. We then measured, using enzyme immunoassay, the concentrations of MMP-9 and of its inhibitor TIMP-1 and evaluated whether airflow limitation may be associated with an imbalance between these enzymes. MMP-9 and TIMP-1 concentrations were greater in sputum of patients with asthma and chronic bronchitis than in control subjects. The molar ratio between MMP-9 and TIMP-1 was lower in asthmatics and chronic bronchitics than in control subjects, and positively correlated with FEV1 values. In asthma, MMP-9 levels were significantly correlated with the number of macrophages and neutrophils. This study shows that airway inflammation in asthma and chronic bronchitis is associated with an imbalance between MMP-9 and TIMP-1 which may have a role in the pathogenesis of ECM remodeling and airflow obstruction.

Increased levels of elastase and alpha1-antitrypsin in sputum of asthmatic patients.

Asthma and chronic bronchitis are inflammatory diseases associated with remodeling of the extracellular matrix (ECM). Elastin, a major component of the ECM in the airways, has been previously found to be disrupted in asthma and chronic bronchitis. This study was aimed at evaluating whether elastin disruption might be associated with an imbalance between elastase (active and total) and alpha1-proteinase inhibitor (alpha1-PI), the main inhibitor of elastase. We measured elastase and alpha1-PI in induced sputum obtained from 16 control subjects, 10 healthy smokers, 19 asthmatic patients, and 10 chronic bronchitis patients. We also assessed the possible origin of elastase, evaluating its levels in sputum with reference to differential cell counts. We found that in induced sputum obtained from asthmatic and chronic bronchitis patients, the levels of both total and active elastase were significantly increased as compared with those of control subjects and healthy smokers and were significantly correlated with the percentage of neutrophils. In addition, in asthma and chronic bronchitis patients, the levels of active and total elastase were inversely correlated with the degree of airway obstruction as assessed from FEV1 values. This study shows that airway inflammation in asthma and chronic bronchitis is associated with high levels of active elastase, which may play a role in the pathogenesis of airway remodeling.

Urinary leukotriene E4 in the assessment of nocturnal asthma.

BACKGROUND: Urinary leukotriene E4 (LTE4) is a marker of the body's production of cysteinyl LTs, important mediators of airway inflammation. The role of the latter in nocturnal asthma is a topic of increasing interest. OBJECTIVE: This investigation was aimed at determining whether nighttime attacks are associated with increased release of LTs, expressed by urinary LTE4, and the relationship between the two phenomena. METHODS: Three groups were studied: group A, seven control subjects; group B, nine asthmatic patients without nocturnal attacks; and group C, nine asthmatic patients with a comparable daytime FEV1 but who were experiencing nocturnal exacerbations (morning dips in peak expiratory flow greater than 20%). Urine was collected over 24 hours in three samples (9:00 AM to 3:00 PM; 3:00 PM to 9:00 PM; and 9:00 PM to 9:00 AM). LTE4 was measured by high-performance liquid chromatography and radioimmunoassay and expressed as nanograms per millimole of creatinine. RESULTS: No significant differences between urinary LTE4 were noticed within groups A and B. Conversely, in group C urinary LTE4 at night (geometric mean with 95% confidence interval; 35.16 with 28.77-42.85) was significantly higher than that of the other samples (respectively 23.12 with 17.78-30.06, p less than 0.05; and 25.18 with 21.03-30.13, p less than 0.02); it was also significantly higher than in all the samples of other groups. A significant (p less than 0.02) linear correlation was observed between morning dip in peak expiratory flow and the log urinary LTE4 in the nocturnal sample. CONCLUSION: These results indicate the role of LTs in nocturnal asthma and suggest that urinary LTE4 may be a useful marker of this condition.