Mechanisms of bradykinin-induced contraction in human fetal lung fibroblasts.

Bradykinin (BK) induces fibroblast contraction but the structural changes and intracellular mechanisms involved have not been completely explored. We stimulated HFL-1 fibroblasts with BK to assess: 1) fibroblast contractility; 2) the role of α-smooth muscle actin (SMA) in contraction by small interfering RNA (siRNA); 3) α-SMA protein expression; 4) α-SMA and F-actin structure; 5) intracellular calcium concentration ([Ca(2+)](i)); and 6) phosphorylated myosin light-chain (pMLC) and MLC kinase (MLCK) expression. BK triggered concentration- and time-dependent fibroblast gel contraction in conjunction with α-SMA over expression, but not in α-SMA-siRNA-treated cells. BK also increased α-SMA(+) and F-actin(+) cell number and stress fibre polymerisation (detectable at 5-60 min). These BK-induced changes were associated with an increase in [Ca(2+)](i), which peaked within 15 s, and activation of pMLC, which was detectable at 5-60 min. No MLCK content modification was observed. The different manifestations of the BK-induced fibroblast activation were downregulated at different levels (25-100%) by HOE140, a specific BK B2 receptor (B2R) antagonist and by the Ca(2+) chelator, EGTA. Thus, BK-induced fibroblast contraction, associated with differentiation into α-SMA(+) myofibroblasts, is mediated through the activation of the B2R and involves the Ca(2+)/calmodulin pMLC-dependent pathway.

Clinical assessment of asthma severity partially corresponds to sputum eosinophilic airway inflammation.

In the aim to evaluate the relationship between sputum eosinophil percentages and eosinophil cationic protein (ECP) concentrations, as markers of airway inflammation, and different Levels of asthma severity, we examined 223 patients consecutively observed in our asthma clinic. Diagnosis of asthma was made according to internationally accepted criteria. Asthma severity was evaluated according to frequency of symptoms, FEV1, peak expiratory flow variability and level of asthma treatment needed to control asthma. Spontaneous or induced sputum was collected. Adequate sputum samples were obtained in 68 untreated subjects and in 117 subjects regularly treated with ICS. A control group of 14 normal subjects was also examined. In untreated subjects, mild intermittent asthmatics showed a lower sputum eosinophil percentage in comparison with other groups of asthma severity, while no difference in ECP levels was detected. In treated subjects, severe asthmatics showed higher levels of sputum eosinophils and ECP in comparison with other groups of asthma severity. Mild persistent and moderate persistent patients did not differ for sputum eosinophils or ECP in both untreated and treated subjects. Controls were significantly different from all groups of untreated and treated asthmatics. In conclusion, the assessment of asthma severity according to clinical and functional findings only partially corresponds to the severity of eosinophilic airway inflammation as assessed by induced sputum analysis.

Gamma radiation inhibits fibroblast-mediated collagen gel retraction.

Radiation exposure is known to impair healing in irradiated areas. Fibroblasts play a major role in the production and modification of extracellular matrix in wound repair. Since one important aspect of wound repair is the contraction of the wound, this study investigated the effects of radiation on the ability of fibroblasts to mediate collagen gel contraction in an in vitro model of wound retraction. After irradiation, the cells were detached and suspended in a solution of rat tail tendon collagen. Radiation exposure decreased retraction, and this effect was dose dependent. In order to define the mechanism of reduced gel retraction, we investigated alpha2beta1 cell surface integrin and fibronectin, which are thought to mediate contraction, and prostaglandin E2 (PGE2), which is known to inhibit this process. PGE2 release increased dose responsively following radiation. The cyclooxygenase inhibitor indomethacin could partially restore the contractile activity of irradiated fibroblasts. Fibronectin production in gel culture showed a significant decrease. In contrast, there was no decrease in alpha2beta1 integrin expression in radiated cells. In conclusion, radiation decreases fibroblast-mediated gel contraction. Increased PGE2 production and decreased fibronectin production by irradiated fibroblasts may contribute to this effect and may be in part responsible for poor healing of radiated tissue.

Fifty microg b.i.d. of inhaled fluticasone propionate (FP) are effective in stable asthmatics previously treated with a higher dose of FP.

Twenty-seven subjects with moderate asthma at the time of diagnosis, well controlled under regular fluticasone propionate (FP) (250 microg b.i.d.) for 6 months at least, were randomized to receive in double-blind fashion: FP 125 microg b.i.d. (Group 1) or FP 50 microg b.i.d. (Group 2) or placebo (Group 3) for 3 months or until symptom recurrence. Daily symptom score and peak expiratory flow were monitored. At the beginning and at the end of the study subjects underwent methacholine challenge and sputum induction. Recurrence of symptoms occurred shortly after randomization in all subjects receiving placebo. None from Group 1 or 2 experienced symptom recurrence during the study. No significant difference in clinical and functional data, and in sputum eosinophil percentages was observed between the beginning and the end of the study in both Groups 1 and 2. Subjects from Group 3 showed a significant increase of sputum eosinophils (P<0.05) and a significant decrease in provocative dose of methacholine (P<0.05) when asthma symptoms recurred. Therefore, very low doses of FP (50 microg b.i.d.) are effective in maintaining for 3 months a good control of the disease in asthmatics already stable under high-dose fluticasone, considering both clinical and functional outcomes and markers of airway inflammation.

Quality evaluation of samples obtained by spontaneous or induced sputum: comparison between two methods of processing and relationship with clinical and functional findings.

The aim of the study was to assess, on a large group of spontaneous or induced sputum samples, the difference in quality between slides processed by two different methods, and the relationship between quality assessment and some clinical and functional characteristics of the examined subjects. We examined 631 sputum samples obtained from 337 subjects with proven (n = 291) or suspected bronchial asthma. Of these, 467 samples were processed using the whole-sample method (Group I), while 164 samples were processed using the plug method (Group II). Salivary contamination, cell distribution on the slide, and cell borders were evaluated, and samples were classified as inadequate, adequate, or good. Inadequate samples were equally represented in both groups, while good samples were represented more in Group II. No significant difference in most clinical and functional findings was observed between the different quality categories of both groups. A higher proportion of inadequate samples was observed in Group I samples spontaneously collected. Mild intermittent asthmatics produced a better quality of slides in comparison with other groups of asthma severity. In conclusion, sputum quality partially depends on the different methods of sputum collection and/or processing, although the percentage of inadequate samples is similar for the two methods of processing. Sputum quality is only marginally affected by clinical and functional characteristics of asthma, or by asthma severity.

Comparison of anti-inflammatory and clinical effects of beclomethasone dipropionate and salmeterol in moderate asthma.

Inhaled corticosteroids and long-acting beta2-agonists effectively control asthma symptoms and improve airway function. The effects of beclomethasone were compared with those of salmeterol on markers of eosinophilic inflammation in induced sputum in steroid-naive asthmatic subjects with moderate asthma. Fifteen moderate asthmatics were treated with either beclomethasone dipropionate (500 microg b.i.d.) or salmeterol (50 microg b.i.d.) for 4 weeks, according to a randomised, double-blind, parallel-group study design. All patients underwent spirometry, methacholine test, sputum induction, and blood sampling before and after 2 and 4 weeks of treatment. They also recorded daily symptoms and peak expiratory flow (PEF). Sputum eosinophils, eosinophil cationic protein (ECP) and eosinophil protein X (EPX), and blood eosinophils, as well as the forced expiratory volume in one second (FEV1) and morning PEF, significantly improved after beclomethasone but not after salmeterol. PEF variability, the symptom score and rescue beta2-agonist use significantly improved after both treatments, although the improvement in the symptom score tended to be greater after beclomethasone. After 2 and 4 weeks of beclomethasone treatment, both serum ECP and EPX decreased. With salmeterol, only serum EPX decreased, after 4 weeks. Bronchial hyperresponsiveness to methacholine did not change after either treatment. The authors conclude that beclomethasone, but not salmeterol, substantially improves airway inflammation in asthma. Beclomethasone also had an overall greater clinical effect, although the improvement in symptoms and peak expiratory flow variability was similar after both treatments.

Budesonide reduces neutrophilic but not functional airway response to ozone in mild asthmatics.

Ambient ozone concentration is related to asthma exacerbation, but few findings are available regarding the effects of pharmacologic asthma treatment on this relationship. The purpose of this study was to investigate whether inhaled corticosteroids inhibit ozone-induced airway neutrophilic inflammation, as detected in induced sputum, and reduce functional response to ozone exposure. Eleven subjects with mild persistent asthma were exposed for 2 h, on separate days, to 0.27 ppm ozone and to air in random order, before and after 4 wk of treatment with budesonide (400 microg twice daily). Before exposure, 1 and 2 h after the beginning of exposure, and 6 h after the end of exposure, pulmonary function was measured, and a total symptom score questionnaire was completed; 6 h after exposure, sputum was induced with hypertonic saline. Budesonide treatment did not inhibit the functional response to ozone exposure, as determined by reduction in FEV(1) and increase in total symptom score, but it significantly blunted the increase in the percentage of sputum neutrophils and interleukin-8 concentrations in the supernatant (p < 0.05). Therefore, 4 wk of inhaled budesonide blunted the airway neutrophilic inflammatory response but did not prevent the functional impairment of the airways after ozone exposure.

Effects of interferons alpha and gamma on cytokine production and phenotypic pattern of human bronchial epithelial cells.

Human bronchial epithelial cells are involved in airway immune mechanisms through secretion of cytokines and through cell-cell contacts with immunocompetent cells. The aim of our study was to assess the ability of interferon (IFN) alpha and gamma alone and in combination to modulate human bronchial epithelial cell (HBECs) release of the inflammatory cytokines IL-8 and IL-6 and fibronectin and to induce the surface expression of HLA-DR and ICAM-1 molecules involved in immune interactions with other cells. HBECs spontaneously secreted a limited amount of IL-8, which was significantly increased by IFN gamma. IFN alpha inhibited IFN gamma stimulated IL-8 secretion in a concentration-dependent manner. Further, IFN gamma induced IL-6 and fibronectin secretion, and this was also inhibited by IFN alpha. The expression of HLA-DR antigens was significantly increased by IFN gamma and partially inhibited by co-stimulation with IFN alpha. In contrast, IFN gamma also induced ICAM-1 expression by HBECs but co-stimulation with IFN alpha had no significant effect on the expression of this surface antigen. IFN alpha modulation of HBEC functions does not seem to be restricted to IFN gamma stimulation since either stimulatory or inhibitory effects of INF alpha on IL-8 production have been found in pilot experiments using IL-1 beta, TNF alpha, and TGF beta as stimuli. In summary, IFN-gamma induces a number of responses in HBECs including increased secretion of IL-6, IL-8 and fibronectin and increased expression of HLA-DR and ICAM-1. IFN alpha can inhibit all these except expression of ICAM-1 which is unaffected. IFN alpha can also interact with other inflammatory cytokines, but whether the effects are inhibitory or augmentive depends on the cytokines.

ICAM-1-independent adhesion of neutrophils to phorbol ester-stimulated human airway epithelial cells.

Intercellular adhesion molecule-1 (ICAM-1) is the only inducible adhesion receptor for neutrophils identified in bronchial epithelial cells. We stimulated human airway epithelial cells with various agonists to evaluate whether ICAM-1-independent adhesion mechanisms could be elicited. Phorbol 12-myristate 13-acetate (PMA) stimulation of cells of the alveolar cell line A549 caused a rapid, significant increase in neutrophil adhesion from 11 +/- 3 to 49 +/- 7% (SE). A significant increase from 17 +/- 4 to 39 +/- 6% was also observed for neutrophil adhesion to PMA-stimulated human bronchial epithelial cells in primary culture. Although ICAM-1 expression was upregulated by PMA at late time points, it was not affected at 10 min when neutrophil adhesion was already clearly enhanced. Antibodies to ICAM-1 had no effect on neutrophil adhesion. In contrast, antibodies to the leukocyte integrin beta-chain CD18 totally inhibited the adhesion of neutrophils to PMA-stimulated epithelial cells. These results demonstrate that PMA stimulation of human airway epithelial cells causes an increase in neutrophil adhesion that is not dependent on ICAM-1 upregulation.

Effect of a single dose of salmeterol on the increase in airway eosinophils induced by allergen challenge in asthmatic subjects.

BACKGROUND: The long acting beta2 agonist salmeterol is very effective in preventing asthmatic responses to specific stimuli, and this effect could theoretically be due to some anti-inflammatory property in addition to bronchodilator property. METHODS: The protective effect of a single dose of salmeterol (50 microg) on allergen induced early and late responses and on the associated airway inflammation was investigated in a double blind, placebo controlled, crossover study in 11 atopic asthmatic subjects. Eosinophil percentages and concentrations of eosinophil cationic protein (ECP) in peripheral blood and in hypertonic saline induced sputum were measured 24 hours after allergen inhalation. RESULTS: Salmeterol effectively inhibited both early and late asthmatic responses in comparison with placebo. Salmeterol also inhibited the increase in the percentage of eosinophils in the sputum 24 hours after allergen inhalation (median (range) baseline 6% (1-36), after placebo 31% (5-75), after salmeterol 12% (1-63)). However, the increase in both sputum and serum ECP concentrations 24 hours after allergen challenge was not affected by pretreatment with salmeterol. CONCLUSIONS: A single dose of salmeterol inhibits the allergen induced airway responses and the increase in sputum eosinophils after allergen challenge.

Salbutamol pretreatment does not change eosinophil percentage and eosinophilic cationic protein concentration in hypertonic saline-induced sputum in asthmatic subjects.

BACKGROUND: Sputum induction by inhalation of hypertonic saline (HS) is usually preceded by beta2-agonist pretreatment, to prevent severe bronchoconstriction. OBJECTIVE: To evaluate whether salbutamol pretreatment may influence cell counts and concentrations of soluble mediators in induced sputum. METHODS: We studied 22 patients who randomly underwent HS sputum induction after pretreatment with either 200 microg salbutamol or placebo. Sputum was induced by means of HS inhalation (3, 4, 5% NaCl, 10 min each), measuring FEV1 every 5 min until it fell >/= 20% from baseline. Collected sputum was diluted 1 : 1 with 0.1% DTT, incubated at 37 degrees C for 20 min, and total and differential cell counts were measured. ECP and histamine levels were measured in the supernatant. RESULTS: Sputum volume, percentages of inflammatory cells, squamous cell counts and quality of the slides were not different after the two pretreatments, while sputum total inflammatory cells after salbutamol tended to be higher than after placebo (8.3 [1-41] 10(6) vs 6.3[0.2-40] x10(6); P = 0.09). Eosinophilic cationic protein (ECP) did not significantly change (260 [8-900] microg/L after salbutamol vs 200 [8-800] microg/L, n = 19), while histamine levels tended to be lower after salbutamol (140.9 [39.9-236.5] nm) than after placebo (190.4 [72. 2-322.6] nm, P = 0.09, n = 17). The airway response to HS inhalation was significantly greater after placebo and the duration of the test was significantly different (median: 15 min after placebo and 30 min after salbutamol). Similar results were obtained when patients who differed for more than 15 min in the duration of HS-inhalation in the two tests were selected (n = 11). CONCLUSION: Salbutamol pretreatment reduces the severity of bronchoconstriction induced by HS inhalation without significantly affecting the percentages of inflammatory cells and the levels of soluble mediators in induced sputum.

Airway inflammatory response to ozone in subjects with different asthma severity.

The aim of this study was to evaluate whether ozone exposure induces a similar airway inflammatory response in subjects with different degrees of asthma severity. Two groups of asthmatic subjects were studied: seven with intermittent mild asthma not requiring regular treatment (group A); and seven with persistent mild asthma requiring regular treatment with inhaled corticosteroids and long-acting beta2-agonists (group B). All subjects were exposed, in a randomized cross-over design, to air or O3 (0.26 parts per million (ppm) for 2 h with intermittent exercise); subjects in group B withdrew from regular treatment 72 h before each exposure. Before the exposure, and 1 and 2 h after the beginning of the exposure they performed a pulmonary function test, and a questionnaire was completed to obtain a total symptom score (TSS). Six hours after the end of the exposure, hypertonic saline (HS) sputum induction was conducted. Sputum cell percentages, eosinophil cationic protein (ECP) and interleukin (IL)-8 concentrations in the sputum supernatant were measured. TSS significantly increased and forced vital capacity (FVC) and forced expiratory volume in one second (FEV1) significantly decreased after O3 exposure in comparison with air exposure in group A, whereas no changes were observed in group B except for a significant decrement of FEV1 2 h after the beginning of O3 exposure. Sputum neutrophil percentage was significantly higher after O3 exposure than after air exposure in both groups (Group A: 70.2% (28-87) versus 26.6% (8.6-73.2); Group B: 62.1% (25-82.4) versus 27.9% (14.4-54)). IL-8 was higher in sputum supernatant collected 6 h after O3 exposure than after air, only in group A. No change due to O3 has been found in sputum eosinophil percentage and ECP concentration in both groups. In conclusion, the degree of airway response to a short-term exposure to ozone is different in subjects with asthma of different severity. The available data do not allow elucidation of whether this difference depends on the severity of the disease or on the regular anti-inflammatory treatment.

Comparison between eosinophilic markers in induced sputum and blood in asthmatic patients.

BACKGROUND: The usefulness and safety of the analysis of blood inflammatory markers in asthma are widely recognized. Recently, the analysis of induced sputum has been proposed as a safe, non-invasive tool in the study of airway inflammation in asthma. OBJECTIVE: Our aim was to test whether sputum analysis is more useful than blood analysis in the evaluation of airway inflammation in untreated and treated asthmatic patients. METHODS: Twelve untreated patients with mild to moderate asthma underwent a methacholine challenge test, sputum induction and blood sampling. A group of 14 normal subjects was also evaluated for baseline comparison. The same evaluation was repeated after 3 months of budesonide treatment. Before and after treatment, we tested the relationship of eosinophilic markers in induced sputum and blood with clinical and functional data. We also compared eosinophilic markers in induced sputum with the same markers in blood. RESULTS: Untreated patients showed a significant relationship between sputum eosinophils and symptom score, and between sputum eosinophilic cationic protein and symptom score, FEV1 and PD20FEV1. No relationship between blood eosinophilic markers and clinical or functional data was observed. In budesonide-treated patients, both sputum and blood eosinophils were significantly lower than in untreated patients, but eosinophil decrease was greater in sputum than in blood. Sputum eosinophilic proteins were also significantly lower in treated patients, whereas serum eosinophilic proteins were low at baseline and remained unchanged after treatment. Sputum eosinophilic markers were lower in normal subjects than in both untreated and treated patients, while blood eosinophils, but not serum eosinophilic cationic protein, were lower in normals than in untreated patients. CONCLUSIONS: The analysis of induced sputum is more useful than the analysis of blood in the evaluation of asthma severity and of the effect of glucocorticoid treatment in patients with mild to moderate asthma.

Leukocyte counts in hypertonic saline-induced sputum in subjects with occupational asthma.

We measured markers of eosinophilic inflammation in the blood and in the sputum induced by hypertonic saline (HS) inhalation of 24 subjects with occupational asthma who were still exposed to high molecular weight compounds (HMWCs, n = 8) or to low molecular weight compounds (LMWCs, n = 16); all subjects were symptomatic and showed bronchial hyperresponsiveness to methacholine at the time of study. Sputum cell counts were also measured in 14 normal subjects and in 24 subjects with non-occupational asthma with asthma severity similar to that of occupational asthmatics. Both occupational and non-occupational asthmatic subjects showed higher neutrophil percentages in HS-induced sputum than normal subjects, asthmatics with LMWC-induced asthma showing the highest values. Eosinophil percentages in HS-induced sputum were higher in non-occupational asthmatics and in asthmatics with HMWC-induced asthma than in normal subjects and in subjects with occupational asthma due to LMWCs. No difference in bronchial responsiveness, peak expiratory flow variability and serum eosinophil cationic protein (ECP) levels were observed among the different asthma groups. Although sputum eosinophil percentages significantly correlated with blood eosinophil percentages, sputum allowed the detection of a higher number of subjects with eosinophilic inflammation than blood. Serum ECP levels were normal in most asthmatic subjects. A significant correlation between sputum eosinophil percentages and bronchial hyperresponsiveness to HS was observed. Despite a similar degree of functional abnormalities, subjects with asthma due to LMWCs and still exposed to the occupational sensitizer showed a lower degree of eosinophilic inflammation and a higher degree of neutrophilic inflammation in the airways than subjects with occupational asthma due to HMWCs or non-occupational asthmatics. Furthermore, sputum eosinophil counts detect, better than blood indices, the degree of airway inflammation in both occupational and non-occupational asthma.

Cigarette smoke extract inhibits fibroblast-mediated collagen gel contraction.

Cigarette smoking, the major cause of pulmonary emphysema, is characterized by destruction of alveolar walls. Because tissue destruction represents a balance between injury and repair, we hypothesized that cigarette smoke exposure may contribute to the development of emphysema through the inhibition of tissue contraction during the repair process. To partially evaluate this hypothesis, we investigated the effects of cigarette smoke extract (CSE) on the ability of cultured fibroblasts to mediate collagen gel contraction in vitro: CSE inhibited fibroblast-mediated gel contraction in a concentration-dependent manner (P < 0.01). Production of prostaglandin E2, a known inhibitor of fibroblast contraction, was unchanged by CSE as was cell surface integrin expression. In contrast, fibronectin production by fibroblasts was inhibited (P < 0.01), and addition of exogenous fibronectin partially restored the contractile activity, thus suggesting at least one mechanism to explain inhibition of gel contraction by CSE. When CSE was treated to remove volatile components, it showed less inhibitory activity on fibroblast-mediated gel contraction. Therefore, we also examined the effects of acrolein and acetaldehyde, two volatile components of cigarette smoke. Inhibition of contraction was observed at 5 microM acrolein and at 0.5 mM acetaldehyde. In conclusion, cigarette smoke inhibited fibroblast-mediated gel contraction, and this inhibition was due, at least in part, to the volatile components of cigarette smoke and may be mediated, at least in part, by a decrease in fibroblast fibronectin production. By inhibition of repair, these smoke components may contribute to the development of pulmonary emphysema.

Relationship between serum heat-stable neutrophil chemotactic activity during early airway reaction to allergen and the pattern of airway response (early versus late reactions) in asthmatic subjects.

In order to evaluate the relationship between allergen-induced heat-stable neutrophil chemotactic activity (HS-NCA) release during early asthmatic reaction (EAR) and the presence of a late asthmatic reaction (LAR), serum HS-NCA was measured at three serum dilutions (1:5, 1:40, 1:200) during EAR induced by allergen in 26 atopic asthmatics, 13 with isolated EAR and 13 with EAR followed by LAR. HS-NCA was measured using a 48-well microchamber with 5-micron-pore-size nitrocellulose filters, using isolated neutrophils from healthy donors and the leading front technique. Subjects with LAR developed EAR after inhalation of a lower dose of allergen than subjects with isolated EAR. Increase in serum HS-NCA during EAR was significantly higher in subjects with isolated EAR than in subjects with EAR plus LAR at the 1:5 dilution, while it was significantly higher in subjects with EAR plus LAR than in the subjects with isolated EAR at the 1:200 dilution; the 1:40 dilution gave similar results in both groups. Changes in serum HS-NCA during EAR significantly correlated with the maximum decrease in forced expiratory volume in 1 s (FEV1) during LAR: a higher decrease in FEV1 during LAR was associated with a lower increase in HS-NCA at the 1:5 dilution (Spearman's rho = 0.43, rho = 0.03), and with a higher increase in NCA at the 1:200 dilution (Spearman's p = -0.46, p = 0.02). These results can be explained by the 'high-dose-inhibition' phenomenon. Assuming that HS-NCA is associated with mast cell degranulation in the airways after allergen challenge, these findings demonstrate that higher mast cell activation during EAR is present in subjects with a subsequent LAR than in subjects with isolated EAR.

Comparison between hypertonic and isotonic saline-induced sputum in the evaluation of airway inflammation in subjects with moderate asthma.

BACKGROUND: Hypertonic saline-induced sputum has recently been used for the evaluation of airway inflammation in asthma. OBJECTIVE: To assess the effect of hypertonicity on airway inflammation. METHODS: We compared the inflammatory cell composition of hypertonic saline-induced sputum with that of isotonic saline-induced sputum in 21 asthmatic subjects and, at baseline and 30 min after each sputum induction, we measured bronchial hyper-responsiveness to methacholine as an indirect marker to detect increased airway inflammation. On two different days, the patients inhaled hypertonic saline (3-5% NaCl) or isotonic saline (0.9% NaCl) for 30 min via an ultrasonic nebulizer, while monitoring FEV1. Sputum was collected for inflammatory cell analysis. RESULTS: There was no difference in inflammatory cell percentages obtained with the two methods. Eosinophils were > 1% in 20 subjects after hypertonic saline and in 16 subjects after isotonic saline, but this difference was not statistically significant. Intraclass correlation coefficients for sputum inflammatory cells obtained with the two methods were +0.642 for eosinophils, +0.644 for neutrophils, +0.544 for lymphocytes and +0.505 for macrophages. Hypertonic saline induced bronchoconstruction in a significantly greater number of subjects than isotonic saline. Also, hypertonic saline increased bronchial responsiveness to methacholine, while isotonic saline did not. CONCLUSION: We conclude that hypertonicity does not affect sputum cell composition, suggesting that inflammatory cells in hypertonic saline-induced sputum are probably preexisting and not acutely recruited in the airways by the hypertonic stimulus. However, the bronchoconstriction and the increase in bronchial hyper-responsiveness after hypertonic saline inhalation may imply the release of inflammatory mediators. This fact must be considered in the evaluation of soluble markers of inflammation in hypertonic saline-induced sputum.

Effect of short-term NO2 exposure on induced sputum in normal, asthmatic and COPD subjects.

The aim of this study was to assess the effects of short-term exposure to low levels of nitrogen dioxide (NO2) on airway inflammation. We studied seven normal, eight mild asthmatic and seven chronic obstructive pulmonary disease (COPD) subjects. All subjects were exposed to air or to 0.3 parts per million (ppm) NO2 for 1 h, with moderate intermittent exercise, on different days and in random order. Before and 2 h after exposure, symptom score and results of pulmonary function tests (PFTs) were assessed. All subjects performed nasal lavage and hypertonic saline (HS) inhalation to collect sputum 2 h after both exposures. Asthmatic subjects had a higher percentage of eosinophils than normal and COPD subjects in HS-induced sputum after air (asthmatics: median 13 (range 0.4-37)%; normals: 0 (range 0-2)%; COPD 1.8 (range 0.1-19)%), whilst COPD patients showed a higher percentage of neutrophils than the two others groups. No significant differences in PFT values or percentages of inflammatory cells were observed in nasal lavage and in HS-induced sputum in normal, asthmatic and COPD subjects after NO2 exposure compared to air exposure, except for a mild decrease in forced expiratory volume in one second (FEV1) 2 h after NO2 exposure in COPD patients. Symptom score showed a mild increase after NO2 exposure both in normal subjects and in COPD patients. We conclude that short-term exposure to 0.3 ppm nitrogen dioxide does not induce an early detectable acute inflammation in proximal airways of normal subjects or of patients with asthma or chronic obstructive pulmonary disease.

Lipopolysaccharide increases fibronectin production and release from cultured lung fibroblasts partially through proteolytic activity.

Fibronectin is a major product of fibroblasts and can mediate diverse functions including wound healing. Chronic bacterial infections are generally associated with a marked decreased in the ability to repair. We therefore hypothesized that bacterial endotoxin, lipopolysaccharide (LPS), might alter fibroblast fibronectin production. LPS augmented fibronectin production by fibroblasts and also stimulated the release of fibronectin from cell layers. An increase in new protein synthesis appeared to account for part of the increased fibronectin, because the inhibitor of protein synthesis, cycloheximide, inhibited the increase in total production of fibronectin. Cycloheximide did not attenuate the increased release of fibronectin into the culture medium. This increased release appeared to be caused, at least in part, by fragmentation of fibronectin by proteases contained in LPS preparations. In this regard all preparations of LPS tested were found to cleave fibronectin. Finally, zymograms indicated that LPS could also cleave gelatin with at least two bands of proteolytic activity but that it did not cleave bovine serum albumin or ovalbumin. These results indicate that the ability of bacterial products to alter fibronectin production and to degrade this macromolecule may account for altered wound repair that occurs with chronic bacterial infection.

Beta-adrenergic agonists attenuate fibroblast-mediated contraction of released collagen gels.

The effects of beta-adrenergic agonists on fibroblast-mediated collagen gel contraction were investigated. beta-Agonists isoproterenol and epinephrine significantly attenuated fibroblast-mediated gel contraction in a concentration-dependent manner, whereas alpha-agonist norepinephrine had no effect. The biologically active form of isoproterenol, (-)-isoproterenol, was 10-fold more effective than the optical isoform, (+)-isoproterenol. beta-Antagonists sotalol and propranolol reversed the attenuation caused by 10(-7) M isoproterenol or epinephrine at the concentration of 10(-7) M or 10(-6) M, but the alpha-antagonist phentolamine did not. However, beta1- or beta2-specificity of these effects is not clear. Isobutyl methylxanthine augmented the effect of isoproterenol and also prolonged the duration. Two reagents which are known to increase intracellular adenosine 3',5'-cyclic monophosphate (cAMP), prostaglandin E2 and dibutyryl adenosine 3',5'-cyclic monophosphate, attenuated gel contraction in a concentration-dependent manner. These data suggest that the fibroblast-mediated collagen gel contraction can be modulated by beta-adrenergic agonists and that the effect depends on cAMP.