Neutrophil serine proteinases and defensins in chronic obstructive pulmonary disease: effects on pulmonary epithelium

Neutrophils have the capacity to accumulate in high numbers in the lung during infection and inflammation. Because they play an important role in host defence against infection, but may also cause tissue injury, these cells are thought to be involved in the pathogenesis of various inflammatory lung disorders, including chronic bronchitis and chronic obstructive pulmonary disease. Neutrophil products that may mediate tissue injury at sites of neutrophil-dominated inflammation include the neutrophil serine proteinases elastase, cathepsin G and proteinase 3, and the nonenzymatic defensins. One of the targets of the neutrophil is the lung epithelium, and in vitro studies have revealed that both the serine proteinases and neutrophil defensins markedly affect the integrity of the epithelial layer, decrease the frequency of ciliary beat, increase the secretion of mucus, and induce the synthesis of epithelium-derived mediators that may influence the amplification and resolution of neutrophil-dominated inflammation. Both neutrophil elastase and defensins induce the release of the neutrophil chemoattractant chemokine interleukin-8 from respiratory epithelial cells. The alpha1-proteinase inhibitor (alpha1-PI) is a well-characterized inhibitor of neutrophil elastase, that also blocks the cytotoxic and stimulatory activity of defensins towards epithelial cells. The elastase inhibitory activity of alpha1-PI is also abrogated by the binding of defensins to this inhibitor. Incubation of epithelial cells with neutrophil defensins in combination with either elastase or cathepsin G resulted in decreased effects on the epithelial cells compared with those observed when the cells were incubated with defensins, elastase or cathepsin G separately. These results suggest that neutrophil defensins and serine proteinases cause injury and stimulate epithelial cells to produce chemokines that attract more neutrophils to the site of inflammation. The effects of neutrophil defensins and serine proteinases on epithelial cells appear to be restricted by proteinase inhibitors and by inhibitory interactions between these sets of neutrophil granule proteins.     

Unopposed neutrophil elastase in bronchoalveolar lavage from transplant recipients with cystic fibrosis

Large numbers of neutrophils with unopposed neutrophil elastase (NE) proteolytic activity are found in lower respiratory tract secretions from most patients with advanced cystic fibrosis (CF). To determine whether antielastase defenses may be overwhelmed in epithelial lining fluid after lung transplantation, we measured NE activity (cleavage of the specific substrate, MeO-Suc-Ala-Ala-Pro-Val-pNA) in bronchoalveolar lavage fluids (BALF) obtained for surveillance or diagnostic purposes at various intervals (1 mo to 7 yr after transplantation) from 52 recipients who had undergone double or bilateral lung transplantation for end-stage CF. Unopposed NE activity was found in BALF from 14 recipients, most of whom also had >= 10(5) colony forming units (cfu) of Pseudomonas aeruginosa in BALF. Ten of the 14 recipients with unopposed NE in bronchoalveolar lavage (BAL) had developed obliterative bronchiolitis (OB), but only 8 of the 38 subjects without unopposed NE activity had OB (p = 0. 002; Fisher exact test). We conclude that antiprotease defenses in lower respiratory tract secretions of CF patients receiving lung allografts are sufficient in the majority of patients to prevent unopposed NE activity. However, the presence of unopposed NE activity in BAL from lung allografts of patients with CF is associated with progressive, irreversible OB and graft failure.     

The role of neutrophils in the pathogenesis of idiopathic pulmonary fibrosis

STUDY OBJECTIVES: The prognostic value of the neutrophil count in BAL fluid (BALF) has been controversial. The role of neutrophils in this inflammatory lung disease, therefore, was evaluated in this study by additional measures. MATERIALS AND METHODS: We performed BAL in 22 patients with idiopathic pulmonary fibrosis (IPF) diagnosed by open lung biopsy specimen. Percent polymorphonuclear leukocyte (PMN) in BALF and absolute neutrophil counts were compared with those of normal nonsmokers. Elastase complexed to alpha-1-proteinase inhibitor (alpha1-PI) in plasma and BALF was measured as a marker of elastase burden, and neutrophil distribution in 22 lung tissues was observed by immunohistochemistry using antineutrophil elastase antibody. RESULTS: Percent PMN and absolute neutrophil counts in BALF did not increase in patients with IPF as compared with normal nonsmokers (n=15); the plasma elastase-alpha1-PI complex value (mean+/-SE) of patients with IPF (668.5+/-112.4 ng/mL) was significantly high as compared with that of normal nonsmokers (130.3+/-21.3, p<0.001). In addition, the BALF elastase-alpha1-PI complex value (mean+/-SE) of patients with IPF was also significantly high (333.1+/-87.0 ng/mg albumin) as compared with that of normal nonsmokers (83.1+/-29.3 ng/mg albumin, p<0.05). Immunohistochemistry demonstrated considerable numbers of neutrophils infiltrating the lung parenchyma in biopsy specimens obtained by open lung biopsy. CONCLUSIONS: These results suggested that although the neutrophil count in BALF could not represent the distribution of neutrophil in the lung, high levels of neutrophil elastase were demonstrated in lung parenchyma and also in both BALF and sera. Therefore, neutrophils might indeed play an important role in the pathogenesis of IPF.     

Neutrophil chemokines in bronchoalveolar lavage fluid and leukocyte-conditioned medium from nonsmokers and smokers

Polymorphonuclear neutrophils (PMN) have been implicated in the pathogenesis of emphysema. The chemokines interleukin-8(IL-8), growth-related oncogene (GRO-alpha) and extractable nuclear antigen (ENA)-78 may be involved in the increased numbers of PMN in smokers' airspaces. The levels of these cytokines in bronchoalveolar lavage fluid (BALF) and bronchoalveolar lavage leukocyte conditioned medium (LCM), along with BALF PMN numbers in 12 smokers who abstained for 12 h (chronic smoking) or continued to smoke until I h before study (acute smoking) and seven nonsmokers were compared. Neutrophils in BALF increased in acute (1.96+/-0.53%, 0.99+/-0.32x10(6) cells) compared with chronic smokers (0.59+/-0.25%, 0.61+/-0.24x10(6) cells, p<0.05 nonsmokers) and nonsmokers (0.79+/-0.29%, 0.05+/-0.01x 10(6) cells, p<0.05). There were no differences in IL-8 or GRO-alpha in BALF between smokers and nonsmokers. ENA-78 levels were lower in smokers (p=0.006). There was no difference in IL-8, GRO-alpha or ENA-78 in LCM from unstimulated cells in smokers versus nonsmokers. After stimulation with lipopolysaccharide (LPS) 10 ng mL(-1), IL-8 release in acute smokers (p=0.04) and GRO-alpha release in smokers (p=0.009) were significantly higher than in nonsmokers. Following stimulation with LPS 100 ng.mL(-1), GRO-alpha release was higher in smokers (p=0.03) and increased further in acute smokers (p=0.02 versus nonsmokers, p=0.04 versus chronic smokers) and ENA-78 release increased in smokers (p=0.02 versus non-smokers). In conclusion, influx of polymorphonuclear neutrophils into smokers' airspaces is an acute phenomenon and neutrophil chemokine release from mixed bronchoalveolar lavage leukocytes is influenced by cigarette smoking and endotoxins.     

Elevated muscle citrate does not reduce carbohydrate utilization during tetanic stimulation

The lysosomal cysteine proteinase cathepsin B is shown to be secreted by ten human colon carcinoma cell lines and to accumulate in culture media as a latent enzyme. The cell lines also secrete a physiological inhibitor of cathepsin B, cystatin C. A significant correlation was found between secretion of the latent enzyme and the inhibitor (r = 0.755, P < 0.01). The aim of the present study was to modulate the respective secretion of the two antagonists to test whether or not latency of cathepsin B was due to the concomitant secretion of the inhibitor. SW480 colon carcinoma cells were treated with the acidotropic agent ammonium chloride, phorbol 12-myristate 13-acetate, and the inflammatory cytokines TGF-beta, TNF-alpha, and IL-1 beta. Ammonium chloride significantly increased latent cathepsin B levels without affecting the constitutive secretion of cystatin C. Phorbol 12-myristate 13-acetate induced a 4- to 5-fold increase in secreted latent cathepsin B, but did not alter significantly the accumulation of cystatin C in media. The cytokines, TGF-beta, TNF-alpha, and IL-1 beta, had no major effect on the expression of these two antagonists. Latent cathepsin B released from human carcinoma cells could be efficiently activated by neutrophil elastase at neutral pH. It is concluded that latent cathepsin B is a true proenzyme rather than an enzyme-inhibitor complex. In addition, our data from neutrophil elastase activation experiments indicate that a proteolytic system for activation of the tumor cell-secreted latent enzyme may exist in vivo.     

Role of neutrophil elastase in allergen-induced airway microvascular leakage in sensitized guinea pigs

To determine the role of neutrophil elastase in allergen-induced airway microvascular leakage, we assessed vascular permeability of guinea pig trachea by measuring the extravasation of Evans blue dye in the circulating blood. Inhalation of ovalbumin (OA) to guinea pigs sensitized with OA caused Evans blue extravasation, indicating an increased microvascular permeability. Pretreatment with ONO-5046 a specific inhibitor of neutrophil elastase, inhibited OA-induced vascular leakage in a dose-dependent manner. Tracheal instillation of human neutrophil elastase likewise increased microvascular permeability, and this effect was almost completely abolished by ONO-5046. Challenge with OA increased the number of neutrophils and neutrophil elastase activity in the bronchoalveolar lavage fluid, and these effects were inhibited by ONO-5046. These results suggest that neutrophil accumulation into the airway and the subsequent release of neutrophil elastase may play a role in the airway microvascular leakage produced by antigen challenge.     

Dyslipidemia and coronary artery disease. Prevalence and treatment in patients referred for coronary arteriography

We report a case with adult respiratory distress syndrome (ARDS) associated with increased levels of squamous cell carcinoma-related antigen (SCC) in the serum and bronchoalveolar lavage fluid (BALF). ARDS was likely induced by ibuprofen, based on the presence of pancytopenia and a weakly positive drug lymphocyte stimulating test (DLST). High serum and BALF levels of interleukin (IL)-8, neutrophil elastase as well as SCC were detected. Corticosteroid therapy resulted in clinical improvement, resolution of pulmonary infiltrates on chest roentgenogram and normalization of serum and BALF levels of IL-8, neutrophil elastase and SCC.     

Epithelial permeability, inflammation, and oxidant stress in the air spaces of smokers

The mechanism responsible for the increased air-space permeability in cigarette smokers is unknown. The aim of this study was to assess the acute and chronic effects of cigarette smoking on epithelial permeability, inflammation, and oxidant stress in the air spaces of smokers. Fourteen cigarette smokers underwent 99mTc-diethylenetriamine pentaacetic acid (99mTc-DTPA) lung scans after abstaining from smoking for 12 h (chronic smoking) and 1 h after smoking two cigarettes (acute smoking). Each smoker also underwent bronchoscopy and bronchoalveolar lavage (BAL) after either chronic (n = 8) or acute smoking (n = 7). Seven nonsmokers also underwent bronchoscopy and BAL. The time to 50% clearance of 99mTc-DTPA (t50) after chronic smoking was 16.7 +/- 1. 3 min (mean +/- SE), and was further reduced after acute smoking to 14.8 +/- 1.0 min (p < 0.01). Neutrophil numbers were increased in bronchoalveolar lavage fluid (BALF) in the acute smoking group as compared with the nonsmokers (p < 0.05). Superoxide release from mixed BAL leukocytes was increased after chronic (p < 0.01) and acute (p < 0.001) smoking, as were thiobarbituric acid-reactive species (TBARS), providing evidence of lipid peroxidation in plasma (chronic, p < 0.05; acute, p < 0.05). Trolox equivalent antioxidant capacity (TEAC) was reduced in plasma (p < 0.001) and increased in BALF (p < 0.05) in both smoking groups. The study therefore showed an acute increase in epithelial permeability and an increase in the number of neutrophils in the air spaces of cigarette smokers concomitant with evidence of increased oxidant stress.     

Secretory leukoprotease inhibitor: partnering alpha 1-proteinase inhibitor to combat pulmonary inflammation

Secretory leukoprotease inhibitor (SLPI) is a low molecular weight serine proteinase inhibitor, notably of neutrophil elastase (NE), which is synthesised and secreted by the pulmonary epithelium. SLPI plays an important role in limiting NE-induced pulmonary inflammation and, significantly, it also possesses anti-HIV activity. SLPI is a significant component of the anti-NE shield in the lung which has different reactivity from, and is therefore complementary to, the anti-NE action of alpha 1-proteinase inhibitor (alpha 1-PI). Inhaled recombinant SLPI (rSLPI) could prove beneficial in partnership with alpha 1-PI in the treatment of a number of inflammatory lung disorders including emphysema, chronic bronchitis, cystic fibrosis, and adult respiratory distress syndrome.     

Alkalinizing water-soluble local anesthetic solutions by addition of cyclodextrin

OBJECTIVE: To correlate indices of airway reactivity to bronchoalveolar lavage (BAL) fluid cytologic features in horses with a recent decline in exercise tolerance. ANIMALS: 20 actively working horses from 2 to 24 years old. PROCEDURE: Bronchoalveolar lavage fluid samples were obtained and analyzed. Forced oscillatory mechanics (1-7 Hz) technique was used for measurements of total respiratory system resistance (RRS), compliance (CRS), and resonant frequency (fres). Changes in RRS (1 Hz) during histamine challenge were used to generate histamine dose-response curves, from which the provocative concentrations that evoked a 75 or 100% increase in baseline RRS (PCRRS75 and PCRRS 100, respectively) were determined. Age, sex, baseline lung mechanics, and BAL cytologic findings were correlated with PCRRS75 and PCRRS100. RESULTS: No horse of the study had clinical signs or history of obstructive pulmonary disease or increased percentage (> 7%) of neutrophils in bronchoalveolar lavage fluid samples. Mean (+/- SEM) RRS, CRS, and fres were 0.67 +/- 0.06 cm of H2O/L/s, 0.52 +/- 0.04 L/cm H2O, and 2.46 +/- 0.02 Hz, respectively. There was no correlation between age or sex, and RRS, CRS, fres, PCRRS75, or PCRRS100. There was a significant correlation (rs = -0.78, P < 0.001) between percentage of BAL fluid mast cells and PCRRS75 or PCRRS100, but correlation with other cell types and indices of airway reactivity were not observed. CONCLUSION: The strong association between mast cell percentage in BAL fluid and airway reactivity in this group suggests that mast cell products may contribute to bronchospasm, airway wall thickening, and/or loss of elastic recoil, which underlie airway hyperreactivity. Alternatively, mast cells may contribute to nonspecific airway reactivity in horses through unknown mechanisms.     

High levels of elastolytic activity in bronchoalveolar lavage fluid from a patient with idiopathic interstitial pneumonia]

A 63-year-old female with acure exacerbution of idiopathic interstitial pneumonia (IIP) showed high levels of elastolytic activity in bronchoalveolar lavage fluid (BALF). She was admitted to our hospital with progressive dyspnea. Arterial blood gas analysis and pulmonary function tests were abnormal. Examination of the bronchoalveolar lavage fluid revealed an increased numbers of neutrophils. Despite the administration of corticosteroids, the patient died of respiratory failure. A high level of elastolytic activity was present in the BALF. Western immunoblot analysis, using an anti-alpha 1-protease inhibitor (PI) antibody, revealed a truncated alpha 1-PI in the BALF. These findings suggest that an imbalance between protease and PI in the lower respiratory tract contribute to lung tissue damage in patients with IIP.     

Induced sputum, bronchoalveolar lavage and blood from mild asthmatics: inflammatory cells, lymphocyte subsets and soluble markers compared

Airway inflammation in asthma can be measured directly by invasive bronchoalveolar lavage (BAL), directly and relatively noninvasively by induced sputum and indirectly from peripheral blood. We compared cellular and fluid phase indices of inflammation in induced sputum, BAL and blood from 11 adults with mild stable asthma. On one day, induced sputum selected from saliva was collected and on the next, blood and BAL. Median results of sputum compared with BAL showed a higher number of nonsquamous cells (53 versus 0.8 x 10(6) cells x mL(-1), p=0.003), more neutrophils (34.3 versus 1.0%, p<0.001), CD4+ and CD19+ T-cells (76.5 versus 54.7%, p=0.01 and 5.2 versus 1.1%, p=0.03, respectively), fewer macrophages (603 versus 95.0%, p=0.002) and markedly higher levels of eosinophil cationic protein (ECP) (264 versus 2.0 microg x L(-1), p<0.001), tryptase (17.6 versus 2.2 UI x L(-1), p<0.001) and fibrinogen (1,400 versus 150 microg x L(-1), p=0.001). Sputum and BAL neutrophils and CD4+ T-cells were strongly correlated. Sputum and BAL differed from blood by having higher proportions of T-cells (94.9 and 98.9% versus 87.7%, p=0.002) and lower proportions of CD19+ T-lymphocytes (p=0.04 and 0.006). Sputum also differed from blood by having higher proportions of CD4+ T-cells (76.5 versus 51.4%, p=0.001), lower proportions of CD8+ cells (24.0 versus 403%, p=0.04) and a higher CD4+/CD8+ ratio (3.3 versus 1.4, p=0.01). We conclude that in mild asthmatics, sputum, bronchoalveolar lavage and blood measure different compartments of inflammation. Induced selected sputum has the advantage over bronchoalveolar lavage of higher density of cell recovery and stronger signal for fluid-phase markers.     

Induction of tumor necrosis factor-alpha and apoptosis in gastric mucosal injury by indomethacin: effect of omeprazole and ebrotidine

BACKGROUND: Inhalation of swine dust causes airway inflammation with influx of inflammatory cells, predominantly neutrophils, into the lungs. A study was undertaken to determine whether or not exposure to swine dust induces release of interleukin 8 (IL-8) into upper and lower airways and how this possible release is related to cellular influx. A further aim was to study the relationship between the inflammatory response and swine dust exposure. METHODS: Thirty one healthy, non-smoking, previously unexposed subjects were exposed to swine dust during three hours work in a swine house. Bronchoalveolar lavage (BAL) was performed two weeks before and 24 hours after the exposure (n = 16). Nasal lavage and acoustic rhinometry were carried out 1-2 hours before and seven hours after the start of the exposure (n = 31). Exposure measurements were performed with personal sampling equipment. RESULTS: The exposure led to 19-fold and 70-fold increases in the neutrophil concentrations in nasal lavage and BAL fluid, respectively (p < 0.001). In BAL, fluid macrophages, lymphocytes and eosinophils increased significantly. The IL-8 levels in BAL fluid increased from < 31.3 ng/l to 63 (43-109) ng/l (median (25-75th percentile), p < 0.001), and in nasal lavage fluid the concentrations increased from 144 (97-227) ng/l to 1064 (864-1437) ng/l (p < 0.001). IL-8 levels showed a significant correlation with the increase in neutrophils in the nasal lavage fluid but not in the BAL fluid. Acoustic rhinometry demonstrated significant swelling of the nasal mucosa. The air concentration of inhalable dust was 23.3 (20.0-29.3) mg/m3, endotoxin 1.3 (1.1-1.4) micrograms/m3, and muramic acid 0.99 (0.78-2.1) microgram/m3. CONCLUSIONS: The concentration of IL-8 increases in BAL fluid and nasal lavage fluid following exposure to swine dust and may be one of the chemoattractants contributing to the recruitment of neutrophils to the nasal cavity and the alveolar space.     

Acute exacerbation following bronchoalveolar lavage in idiopathic interstitial pneumonia

We report two cases of idiopathic interstitial pneumonia (IIP) with acute deterioration after bronchoalveolar lavage (BAL). Case 1 was a 54-year-old woman, and case 2 was a 75-year-old man. Both were diagnosed as having IIP, and hospitalized with complaints of high fever and dyspnea. After BAL, the degree of dyspnea increased. White blood cell count and lactate dehydrogenase were elevated, and PaO2 was decreased. Chest X-ray revealed consolidation of the upper lung fields and reticulo-granular shadows spreading through both lungs. BAL fluid examination showed an elevated neutrophil fraction (case 1: 6.5%, case 2: 35.2%), suggesting respiratory tract infection although bacteria could not be detected. Case 1 died of respiratory failure despite corticosteroid therapy. Autopsy revealed diffuse alveolar damage with focal pneumonia in the right S6 corresponding to the upper lobe consolidation. Case 2 improved after antibiotic therapy. These findings suggest that aspiration of infected fluid during BAL can cause acute exacerbation of IIP. It is important to recognize that the BAL procedure can induce an acute exacerbation of IIP.     

Grain dust-induced airflow obstruction and inflammation of the lower respiratory tract

To investigate the relationship between the physiologic and biologic effects of grain dust inhalation, we exposed 15 nonsmoking, nonasthmatic, nonatopic male grain handlers to buffered saline and aqueous corn dust extract by inhalation challenge in a crossover study. The inhalation challenges to buffered saline and corn dust extract were separated by at least 14 d. Compared with buffered saline, inhalation of corn dust extract resulted in significant airflow obstruction, which was observed within 30 min of exposure and persisted for 5 h. Inhalation of corn dust extract resulted in an acute inflammatory response characterized by higher concentrations of neutrophils (p = 0.001), IL-1 beta (p = 0.001), IL-1RA (p = 0.001), IL-6 (p = 0.001), IL-8 (p = 0.001), and TNF-alpha (p = 0.04) in bronchoalveolar lavage (BAL) fluid. mRNA levels specific for IL-1 beta, IL-1RA, IL-6, and IL-8 from cells present in the BAL fluid were significantly greater after challenge with corn dust extract than after challenge with buffered saline. Importantly, no significant differences were observed in the concentration of lymphocytes or eosinophils in the BAL fluid following inhalation of corn dust extract, and the concentrations of histamine and 15-HETE were similar in BAL fluid after the two challenges. The maximal percentage decrease in FEV1 was significantly associated with the absolute neutrophil concentration in the BAL fluid (p = 0.001), as well as the concentration of TNF-alpha (p = 0.03), IL-1 beta (p = 0.005), IL-1RA (p = 0.001), IL-6 (p = 0.001), and IL-8 (p = 0.001) in the BAL fluid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphology of the lungs and cellular composition of bronchoalveolar lavage in tuberculomas

Morphology of the lungs and bronchoalveolar lavage (BAL) were studied in 27 patients with tuberculomas. The investigators also determined lymphocytic, macrophagal or neutrophil BAL composition regarding lymphocyte, macrophage or neutrophil dominating infiltration of pulmonary tissue outside the sites of specific inflammation. As a result, the activity of the process was assessed by subpopulation composition of lymphocytes in BAL, by morphology of alveolar macrophages in pulmonary tissue; the pattern of inflammatory reaction in the lesion focus was specified.     

Anti-pneumococcal vaccination in elderly persons

To assess the role of some pro-inflammatory cells in inflammatory processes in lung cancer by measuring their respective activation markers in different portions of bronchoalveolar lavage (BAL) fluid. Prospective study in a university hospital. We studied 52 BAL samples, 37 from patients with lung cancer and 15 from a control group, using a radioimmunoassay technique to analyze for tryptase (T), hyaluronic acid (HA) and eosinophil cationic protein (ECP) in separate bronchial and bronchoalveolar samples from BAL fluid. Statistical analysis was performed using the R-SIGMA program. Patients with tumors had significantly higher T and HA levels in BAL fluid than did control patients, in both bronchial and bronchoalveolar portions. Lung cancer patients had higher T and ECP levels in bronchoalveolar portions. Mast cells and fibroblasts, at least, play a part in lung cancer, mainly in the distal portions of the bronchial tree.     

Circulating vascular cell adhesion molecule-1 in pre-eclampsia, gestational hypertension, and normal pregnancy: evidence of selective dysregulation of vascular cell adhesion molecule-1 homeostasis in pre-eclampsia

The potential of DNase I to increase cystic fibrosis sputum elastase activity and lung damage was evaluated. Sputum from CF patients induced little lung hemorrhage when instilled intranasally in C57BL/6 mice. However, sputum treated in vitro by the addition of 1 mg/ml bovine DNase I showed increased neutrophil elastase activity (7.97 +/- 1.56 versus 3.91 +/- 0.62 microM, p < 0.01) and induced marked lung hemorrhage in mice (bronchoalveolar lavage fluid hemoglobin = 192.8 +/- 40.7 versus 44.5 +/- 12.0 microg/ml, p < 0.01). These effects were not observed with DNase I alone in phosphate buffer and were suppressed by the human neutrophil elastase inhibitor methoxysuccinyl-alanyl-alanyl-prolyl-valine-chloromethylketone (MeOSAAPV-CMK). In vivo administration of 2.5 mg aerosolized recombinant human DNase I to patients with CF resulted in a 2.2-fold increase of sputum elastase activity within 1 h of treatment. Elastase levels returned to pre-rhDNase therapy levels 24 h after aerosol treatment. Sputum collected 1 h after rhDNase on 4 separate days from two of six patients in which elastase levels were highest, induced lung hemorrhage when instilled intranasally in mice. We conclude that DNase I therapy of patients with cystic fibrosis can acutely increase the elastase activity of sputum and also its potential to induce hemorrhage in the murine lung.     

New approaches to Pseudomonas aeruginosa lower respiratory tract infections

Since 1973, the occurrence of respiratory tract infections due to P. aeruginosa has increased associated with the development of broad-spectrum penicillins. A clinical entity, diffuse panbronchiolitis (DPB) is a representative disease of chronic P. aeruginosa infections in Japan. In this paper, recent advances of research on pathogenesis and treatments of chronic P. aeruginosa lower respiratory tract infections in our department are reported. We examined sputum from patients with chronic P. aeruginosa infections under the electron microscope. Mucoid type of microcolonies were observed with fibrous matrix of exopolysaccharide. Neutrophils were found to be partially surrounding the microcolony in an attempt to defense. Debris was formed mainly by the destruction of the neutrophils. Most neutrophils were found full of phagocytized debris. These data support that instead of phagocytizing bacteria, neutrophils phagocytized debris and bacteria were not completely eradicated. This might be a factor in the pathogenesis of persistent colonization of P. aeruginosa. In the airways of patients with chronic airway diseases (CAD), neutrophils enhance the recruitment of more neutrophils through the production of neutrophil chemotactic factors such as interleukin-8 (IL-8) and LTB4, perpetuating a cycle of inflammation in the lung. We demonstrated increased levels of IL-8, a chemotactic cytokine, in bronchoalveolar lavage (BAL) fluid from patients with CAD associated with P. aeruginosa infections. We also documented a significant correlation between neutrophil numbers and IL-8 levels or IL-1 beta levels or neutrophil elastase levels in BAL fluids from patients with CAD. By immunohistochemical studies and in vitro data, three major sources of IL-8 in the airways of CAD patients were found to be alveolar macrophages, bronchial epithelial cells, and migrated neutrophils. In Japan, the clinical effectiveness of oral erythromycin (EM) for CAD, including DPB seems to be established, but its pharmacological mechanism remains unclear. In addition, we found a marked decrease of IL-8 levels in BAL fluid from two patients with CAD after treatment with EM. Therefore, we postulated that EM inhibited IL-8 production by stimulated respiratory cells. EM and Roxythromycin, suppressed IL-8 production in Pseudomonas-stimulated neutrophils in a dose-dependent manner. 1 alpha, 25-dihydroxy vitamin D3 also inhibited neutrophil-derived IL-8. Our data encourage the development of new anti-IL-8 agents against persistent P. aeruginosa lower respiratory tract infections.     

Estimation of relative economic value for herd life of dairy cattle from profile equations

The phagocytic capability afforded by neutrophil influx into the lungs is essential to ward off invading bacteria. The objective of this study was to evaluate the effect of prior neutrophil recruitment induced by alveolar instillation of endotoxin (LPS, 200 micrograms/kg) 16 h before a pulmonary infection caused by instillation of live Pseudomonas aeruginosa ([PYO]: 1.5 x 10(8) colony-forming units [cfu]/kg) in rats. A first series of experiments showed that lipopolysaccharide (LPS) instillation induced recruitment of alveolar neutrophils that were capable, ex vivo, of elastase exocytosis, reactive oxygen species secretion, and PYO killing. In a second set of experiments, LPS followed by PYO was compared with PYO alone (n = 11 surviving rats in each group). Parameters were studied 24 h after the bacterial challenge. As compared with PYO alone, pretreatment with LPS followed by PYO was associated with decreased mortality (0% versus 54%, p < 0.05), decreased protein leakage into bronchoalveolar lavage (BAL) fluid (1.8 +/- 0.4 versus 13.5 +/- 2.2 mg/ml, p < 0.001), and improved bacterial clearance from BAL (4.0 +/- 1.4 x 10(2) versus 1.2 +/- 0.5 x 10(4) cfu/ml, p < 0.05) and from pulmonary parenchyma (8.5 +/- 6.4 x 10(5) versus 1.9 +/- 0.8 x 10(7) cfu/ml, p < 0.05). We conclude that prior alveolar endotoxin instillation induces local recruitment of functionally active neutrophils, and that this is associated with resistance to subsequent experimental pneumonia.