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alpha1-Antitrypsin (AAT) deficiency predisposes to bronchitis and emphysema associated with neutrophilic airway inflammation. The efficacy of augmentation therapy has not been proven clinically or by demonstrating an effect on airway inflammation. We treated 12 patients with four infusions of Prolastin (60 mg/kg) at weekly intervals and monitored both the serum and secretion concentrations of AAT as well as markers of neutrophilic inflammation, including myeloperoxidase, elastase, and the neutrophil chemoattractants interleukin-8 and leukotriene B(4). Serum AAT rose and was maintained above the protective threshold. In addition, AAT concentrations in the sputum rose from a mean of 0.17 microM (SEM +/- 0.04) before therapy to concentrations similar to nondeficient subjects (0.43 +/- 0.12) 1 week after the first infusion (p < 0.01). This was associated with a reduction in elastase activity (p < 0.002) and the chemoattractant leukotriene B(4) (p < 0.02), which fell from a median baseline value of 13.46 nM (range, 4.17-55.00) to 8.62 nM (4.23-21.59) the day following the last infusion. Although median values for myeloperoxidase and interleukin-8 also fell, the changes failed to achieve statistical significance. In summary, short-term therapy with AAT increased lung secretion concentrations and was associated with a fall in leukotriene B(4), which is thought to be central to the airway inflammation of AAT deficiency.

Patients with homozygous (PiZ) alpha(1)-antitrypsin (AAT) deficiency have not only low baseline serum AAT levels (approximately 10 to 15% normal) but also an attenuated acute phase response. They are susceptible to the development of premature emphysema but may also be particularly susceptible to lung damage during bacterial exacerbations when there will be a significant neutrophil influx. The purposes of the present study were to assess the inflammatory nature of acute bacterial exacerbations of chronic obstructive pulmonary disease (COPD) in subjects with AAT deficiency, to compare this with COPD patients without deficiency, and to monitor the inflammatory process and its resolution following appropriate antibacterial therapy. At the start of the exacerbation, patients with AAT deficiency had lower sputum AAT (p < 0.001) and secretory leukoprotease inhibitor (SLPI; p = 0.02) with higher elastase activity (p = 0.02) compared with COPD patients without deficiency. Both groups had a comparable acute phase response as assessed by C-reactive protein (CRP) but the AAT-deficient patients had a minimal rise in serum AAT (to < 6 microM). After treatment with antibiotics, in patients with AAT deficiency, there were significant changes in many sputum proteins including a rise in SLPI levels, and a reduction in myeloperoxidase (MPO) and elastase activity (p < 0. 005 for all measures); the sputum chemoattractants interleukin-8 (IL-8) and leukotriene B(4) (LTB(4)) fell (p < 0.01), and protein leak (sputum/serum albumin ratio) became lower (p < 0.01). The changes were rapid and within 3 d of the commencement of antibiotic therapy the biochemical markers had decreased significantly, but took a variable time thereafter to return to baseline values. In conclusion, patients with AAT deficiency had evidence of increased elastase activity at the start of the exacerbation when compared with nondeficient COPD patients which probably reflects a deficient antiproteinase screen (lower sputum AAT and SLPI). The increased bronchial inflammation at presentation resolved rapidly with 14 d of antibiotic therapy.

Inhaled corticosteroids are widely prescribed for the treatment of stable chronic obstructive pulmonary disease (COPD), despite lack of proven efficacy. Because COPD involves airway inflammation and probable protease-antiprotease imbalance, we examined the effect of high dose fluticasone propionate on markers of activity of both pathogenetic mechanisms. Thirteen patients with COPD were treated with fluticasone propionate (500 microg twice a day) for 4 wk, delivered via MDI and spacer, in a double-blind crossover study. There was no clinical benefit in terms of lung function or symptom scores, and induced sputum inflammatory cells, percentage neutrophils, and IL-8 levels were unchanged. Sputum supernatant elastase activity, matrix metalloproteinase (MMP)-1, MMP-9, and the antiproteases secretory leukoprotease inhibitor (SLPI) and tissue inhibitor of metalloproteinase (TIMP)-1 were similarly unaffected by treatment. These results add to previous evidence that inhaled steroids have no anti-inflammatory action in stable COPD. Furthermore, inhaled steroids do not appear to redress the protease-antiprotease imbalance that is thought to be important in the pathogenesis of airway obstruction.

Local immunoglobulins play a key role in host defense against lung infection. We investigated the pattern of evolution of bronchial albumin, IgA, and IgG levels in ventilated ICU patients in relation to nosocomial pneumonia. Immunocompetent, critically ill patients underwent serial blood and bronchial protein determinations on Day 1 (intubation day), and on Days 3, 7, 10, and 14. The variations in proteins levels were compared with corresponding Day 1 values in the whole population, and between patients who developed lung infections (Group A) and the remaining population (Group B). Forty-four patients were included into the study. In the whole population, when compared with the baseline value, bronchial IgA/albumin ratio increased significantly (Day 3, +58%, p = 0.04); Day 14, +171%, p < 0.01), but serum IgA/albumin and serum and bronchial IgG/albumin ratios did not change significantly. In Group A, the increase in the IgA/albumin ratio was less than in Group B (Day 3, +15% versus +87%, p = 0.04; Day 14, +29% versus +210%, p < 0.01). No significant differences were observed between the two groups for bronchial and plasma albumin and IgG levels and for bronchial polymorphonuclear elastase levels. Bronchial IgA production was enhanced in ventilated patients. A reduction in this enhanced bronchial IgA production might account for the development of nosocomial pneumonia.

The effects of fluticasone propionate (FP) on sputum chemotactic activity, elastase inhibitory potential, albumin concentrations, and peripheral neutrophil function were studied in a group of patients with clinically stable, smoking-related chronic bronchitis and emphysema. Seventeen patients (50 to 75 yr of age) were entered into a double-blind, placebo-controlled study of 1.5 mg inhaled FP/d for 8 wk. Following treatment with FP the chemotactic activity of the sputum sol phase was lower than the corresponding values for the placebo group (p < 0.01). Values fell from a mean of 21.75 (+/- 1.58) during the run-in period to 18.37 (+/- 1.46; p < 0.01) after 4 wk and 17.63 (+/- 1.86; p < 0.05) after 8 wk treatment returning to 22.08 (+/- 1.26) cell/field after the washout period. The neutrophil elastase inhibitory capacity of the sputum sol phase increased (p < 0.025) with treatment from a mean of 0.177 microM elastase inhibited/L (+/- 0.05) pretreatment to 0.413 microM (+/- 0.054) after 4 wk and 0.415 microM (+/- 0.054) after 8 wk returning to 0.270 microM (+/- 0.07) after the washout period. Treatment with FP did not result in a change in the peripheral neutrophil functions studied or sputum albumin and myeloperoxidase concentrations. The results suggest that FP may play a protective role in these patients through a reduction in the chemotactic activity of lung secretions and potentially a reduction in the recruitment of neutrophils to the lung, and also by directly affecting the proteinase/antiproteinase balance, in favor of antiproteinases, within lung secretions.

Because interleukin 8 (IL-8) is a potent neutrophil chemotactic and activating cytokine, we investigated IL-8 production in relation to neutrophil migration and elastase release in the human lung during unilateral community-acquired pneumonia (CAP). In 17 patients, the local response in the involved lung was compared with that in the contralateral, noninvolved lung, and with the systemic response. Eight healthy volunteers served as controls. IL-8, total neutrophil elastase (NE), free elastase activity, alpha 1-antitrypsin (alpha 1-AT), and total leukocyte and neutrophil counts were evaluated in bronchoalveolar lavage fluids (BALF). Mean IL-8 concentrations in BALF from the involved lungs of the patients were significantly greater than those in BALF from the noninvolved lung or from controls (p < or = 0.001). By contrast, the serum IL-8 concentration was not different in patients and in controls. Total NE and alpha 1-AT concentrations were increased in BALF from the involved lung as compared with the noninvolved lung or controls (p < or = 0.001). The elastase-inhibitory capacity of alpha 1-AT in BALF was impaired in the involved lung of seven of the 14 patients as compared with the controls, leading to free elastase activity in the involved lung of all patients with CAP. Plasma total NE concentrations were significantly greater in the CAP patients than in the controls. IL-8 concentrations in BALF correlated positively with total leukocyte counts, absolute numbers and percentages of neutrophils, total NE concentrations, and free elastase activity. Our results suggest that during unilateral CAP, locally produced IL-8 may trigger neutrophil accumulation and activation, thus contributing to a local elastase/antielastase imbalance within the site of infection.

The purposes of this study were to investigate the effect of our recently developed truncated secretory leukoprotease inhibitor (SLPI) on bleomycin (BLM)-induced lung injury and fibrosis in hamsters, which is widely used as a model of interstitial pulmonary fibrosis, and to assess the possible involvement of neutrophil elastase in the pathogenesis of pulmonary fibrosis. The fibrosis model was prepared by administration of BLM (10 mg/kg) intratracheally to hamsters. The truncated SLPI was administered at a dose 5 or 15 mg/kg intraperitoneally twice a day only for 10 d starting from the time of BLM administration. BLM administration resulted in decreases in body weight and survival rate. Elastase activity in the supernatant of bronchoalveolar lavage (BAL) fluids was increased at 3 and 7 d after BLM administration in parallel with the increase in the number of neutrophils in the fluids. Histopathologically, at 14 and 28 d after BLM administration, diffuse thickening and fibrosis of alveolar walls with marked cell infiltration and severe distortion of alveolar structure, including honeycomb lung, were observed to have occurred. In this model, the decreases in body weight and survival rate and the increase in elastase activity were inhibited by the truncated SLPI dose-dependently, and pulmonary fibrosis was significantly ameliorated by the administration of this shortened form of SLPI. These results suggest that neutrophil elastase may be implicated in the pathogenesis of BLM-induced pulmonary fibrosis and that the truncated SLPI might be a promising therapeutic in the treatment of interstitial pulmonary fibrosis.

In an attempt to further evaluate the role of neutrophil elastase (NE) in the development of emphysema, we examined the immunologic quantity of NE bound to alpha 1-protease inhibitor (PI), the NE inhibitory activity, and the molecular pattern of alpha 1-PI in unconcentrated bronchoalveolar lavage fluid (BALF) supernatant from 36 community-based older volunteers. They were classified into three groups: 10 current smokers with low attenuation areas (LAAs) on the lung computed tomography (CT) scans who were considered to have subclinical emphysema, 13 current smokers who had a comparable smoking history but no LAA, and 13 noncurrent smokers without LAA. The concentration of NE-alpha 1-PI complex was significantly increased in the subjects with subclinical emphysema when compared not only with the noncurrent smokers (0.52 +/- 0.10 versus 0.21 +/- 0.03 SEM micrograms/mg albumin, p < 0.01) but also with the LAA(-) current smokers (0.52 +/- 0.10 versus 0.23 +/- 0.07 SEM micrograms/mg albumin, p < 0.01). NE inhibitory activity measured by a spectrophotometric method using methoxysuccinyl-alanyl-alanyl-prolyl-valyl-paranitroanilide did not show any significant difference between the two groups of current smokers. There was no difference in the pattern or density of native and proteolysed alpha 1-PI bands between the three groups by Western blotting. We conclude that NE-alpha 1-PI complex in BALF is a factor that may differentiate smokers who are potentially developing emphysema from those who are not.

Elastin degradation has been reported to be increased in patients with cystic fibrosis (CF). In order to further explore evidence for elastin degradation in a group of 18 patients with CF with a wide range of disease severity, we used an isotope dilution method to measure urinary desmosine (DES) and isodesmosine (IDES), amino acids derived exclusively from cross-linked elastin, and hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP), amino acids derived exclusively from cross-linked collagen. Urinary DES and IDES (mean +/- SD) were 23.9 +/- 30.7 and 18.5 +/- 22.4 micrograms/g creatinine, respectively, in the patients with CF versus 7.5 +/- 1.7 and 6.8 +/- 1.4 micrograms/g creatinine, respectively, in 10 healthy control subjects (p < 0.001); only two patients with CF had DES values within the control range. The values of urinary HP and LP in the CF group were 54.9 +/- 39.1 and 12.3 +/- 8.6 nmol/mmol creatinine, respectively, versus 24.5 +/- 5.8 and 5.1 +/- 2.7 nmol/mmol creatinine, respectively, in the controls (p < 0.005). Both HP and LP were highly correlated (r = 0.71, p < 0.0001). Patients with CF had active pulmonary inflammation; neutrophils were abundant in the bronchoalveolar lavage fluid of the CF group and correlated with elastase activity measured with methoxysuccinyl Ala-Ala-Pro-Val paranitroanilide (r = 0.61, p < 0.05). Airway neutrophils had decreased expression of the complement receptor CR1 (CR1/CR3 of 0.17 +/- 0.15 versus 1.0 for blood neutrophils), a change known to be caused by uninhibited neutrophil elastase. We conclude that lung elastin is the most likely source of the increased DES and IDES in CF.

We studied the effect of nasal administration of capsaicin in eight patients with allergic rhinitis and eight healthy subjects. We also studied the effect of colchicine, a drug known to inhibit microtubular axonal transport of peptides, on nasal response to capsaicin in these subjects. Colchicine or placebo was administered orally in a double-blind, randomized, crossover manner with a 35 day wash-out interval. Nasal challenge was performed on the last day of each period of treatment, using increasing doses of capsaicin (10(-9) to 3 x 10(-5) mmol). Capsaicin induced a dose-dependent decrease in nasal airflow conductance (active posterior rhinomanometry) (p < 0.002) that was greater in patients with allergic rhinitis (0.40 +/- 0.02 to 0.20 +/- 0.03) than in healthy subjects (0.44 +/- 0.01 to 0.35 +/- 0.02) (p = 0.0001). Capsaicin provoked a greater number of sneezes in patients with allergic rhinitis than in healthy subjects (p < 0.001), but the amount of nasal secretions was similar in these two groups of subjects. In nasal lavage fluid, capsaicin induced an increase in total, epithelial, and neutrophil cell counts in patients with allergic rhinitis (each comparison, p < 0.05), but not in healthy subjects. Capsaicin induced a slight, although not significant, increase in the concentration of elastase in nasal lavage fluid in patients with allergic rhinitis (p = 0.07), but not in healthy subjects. Albumin concentration decreased in nasal lavage fluid in both groups of subjects (p < 0.05). The tendency of capsaicin to increase neutrophil elastase in nasal lavage fluid of patients with allergic rhinitis was not observed after treatment with colchicine.(ABSTRACT TRUNCATED AT 250 WORDS)

We investigated the mechanisms and consequences of neutrophil accumulation in the airspace in 11 patients with systemic sclerosis (SSc) and interstitial lung disease. Seven normal subjects served as controls. We measured total neutrophil elastase burden, elastase activity, and alpha-1-antitrypsin (alpha 1AT) in bronchoalveolar lavage (BAL) fluid and we evaluated the in vitro interleukin-8 (IL-8, a potent chemoattractant for neutrophils) secretion by alveolar macrophages (AM). A mild neutrophil alveolitis was observed in patients when compared with control subjects. Total BAL elastase burden was higher in patients than in control subjects and correlated positively with the percentage of neutrophils in BAL. BAL elastase activity was undetectable in control subjects, but it was detected in all patients but one (mean: 257 +/- 87 mU/L). Spontaneous IL-8 secretion by AM was higher in patients with SSc than in control subjects (518 +/- 115 versus 228 +/- 65 ng/ml, p = 0.04) and positively correlated with the percentage of neutrophils in BAL (r = 0.505). We conclude that (1) the neutrophil could participate in the pathogenesis of SSC lung disease through the release of elastase; (2) the AM could contribute to the influx of neutrophils in the alveolus through the release of IL-8.

The potential role of neutrophil elastase in causing lung damage and exacerbating the inflammatory response in cystic fibrosis (CF) has received considerable attention. Although another potent neutrophil-derived enzyme, collagenase, is implicated in tissue destruction in several interstitial lung disorders, there has been no reference to this enzyme in CF. The objective of this study was to determine whether neutrophil collagenase is present in active form in CF sputum and, if so, whether it is related to disease severity. High levels of active collagenase were detected in sputum from patients with CF, and the majority of the enzyme present was of neutrophil origin. In a group of 16 patients with CF, negative relations between sputum collagenase activity and Shwachman score (r = -0.55, p < 0.05) and FEV1 (r = -0.59, p < 0.02) were noted, indicating an association between high collagenase activity and severity of disease. A positive correlation was observed between sputum collagenase and elastase activity (r = 0.62, p < 0.05). These results suggest that both neutrophil elastase and collagenase may play a significant role in lung destruction in CF.

To determine the extent of airway infection and inflammation in adolescents and adults with cystic fibrosis (CF) who have mild lung disease and are without symptoms of active infection, we performed bronchoalveolar lavage (BAL) on 18 CF patients > or = 12 yr of age who were stable, appeared clinically well, and had mean (+/- SEM) FEV1 of 79 +/- 4% of predicted. We quantitated the bacteria, inflammatory cells, immunoglobulins, and mediators of inflammatory tissue damage in the epithelial lining fluid (ELF) of these patients and in 23 healthy control subjects. All CF patients were found to be infected with Pseudomonas aeruginosa, Staphylococcus aureus, and/or Haemophilus influenzae; no organisms were isolated from the control subjects. The mean number of cells in the ELF was 14 times greater in the CF patients than in the control subjects. Neutrophils constituted 57% of the recovered cells in the CF patients versus 3% in the control subjects, and their concentration was 380 times greater in the CF patients versus the control subjects. IgG, IgA, and IgM were 2.5 to 6 times greater in CF ELF versus that of control subjects. Abundant active elastase was present in the ELF of the CF patients (2.3 +/- 0.9 microM) despite threefold elevated levels of alpha 1-protease inhibitor (alpha 1-PI). No active elastase was detectable in the control subjects. alpha 1-PI was functional in CF as demonstrated by elevated elastase:alpha 1-PI complex (0.045 microM in CF versus 0.002 microM in control subjects). This active elastase caused proteolytic destruction of surface complement receptors on airway neutrophils in situ.(ABSTRACT TRUNCATED AT 250 WORDS)

Exoglycosidases remove peripheral monosaccharides from oligosaccharides and hence are capable of altering respiratory epithelial cell surface carbohydrates. We obtained saliva and tracheal secretions from 34 critically ill patients and saliva from 23 healthy subjects. Compared with the normal subjects, the ill patients had large amounts of mannosidase, fucosidase, hexosaminidase, and sialidase activity. Sialidase increased adherence of several gram-negative bacteria to epithelial cell monolayers and pure glycoproteins. Pretreatment of glycoproteins with some of the patients' saliva samples also increased bacterial adherence to the glycoproteins. We conclude that respiratory tract exoglycosidase activity increases during critical illness. By altering normal cell surface carbohydrates, exoglycosidases may facilitate bacterial adherence and respiratory tract colonization.

Cystic fibrosis (CF) is characterized in the lung by chronic purulent bronchitis culminating in pulmonary insufficiency. There is evidence to suggest that neutrophil elastase (NE) released by neutrophils on the respiratory epithelial surface plays a major role in the pathogenesis of this lung disease. This study sought to determine the age of onset of the chronic neutrophil-dominated inflammation in CF and the consequences to the NE-anti-NE screen on the respiratory epithelial surface of the CF lung. NE and anti-NE defensive molecules were evaluated in respiratory epithelial lining fluid (ELF) in 27 children with stable CF (1 to 18 yr of age). Despite normal antigenic concentrations of alpha 1-antitrypsin (alpha 1AT) and secretory leukoprotease inhibitor (SLPI), 25 of 27 children with CF had neutrophil-dominated inflammation (> 500 neutrophils/microliters ELF). Active NE was found in ELF in 20 of 27 children, including two of four aged 1 yr. Western blot analysis showed the majority of alpha 1AT and SLPI molecules to be complexed and/or degraded. These observations demonstrate that a chronic imbalance of the NE-anti-NE protective screen develops early on the respiratory epithelial surface in persons with CF and is likely well established by 1 yr of age, with resultant potential for lung damage.

Segmental bronchoprovocation (SBP) with allergen was used in an attempt to study eosinophils recruited to the airway 24 h after challenge. Unexpectedly, in the first four patients, neutrophils (rather than eosinophils) were recruited in the bronchoalveolar lavage (BAL) fluids, and we hypothesized that the allergen extracts were contaminated with endotoxin. The extracts used for challenge in the first four patients tested positive for bacterial endotoxin in a limulus amebocyte lysate assay. Rechallenge of one patient from the first group with a comparable dose of an endotoxin-free extract and SBP with endotoxin-free extract in five additional patients resulted in preferential recruitment of eosinophils rather than neutrophils. The number of neutrophils recovered from the challenged segments in the patients challenged with endotoxin-free extract was significantly less than that observed in the first four patients. Taken together, these observations suggest that neutrophil recruitment in the 24-h BAL fluids from the first four patients was probably due to endotoxin contamination of the allergen extract. We caution investigators that endotoxin contamination of allergen extract may alter the cellular inflammation during the late airway response following allergen challenge.

We examined the proteinase-antiproteinase balance in the bronchoalveolar lavage (BAL) fluid from alpha-1-proteinase inhibitor (alpha 1PI)-deficient lung transplant recipients to determine whether they would derive benefit from intravenous augmentation therapy with alpha 1PI. BAL fluid from 11 alpha 1PI-deficient lung transplant recipients and eight control subjects was assayed for free neutrophil elastase activity, immunoreactive alpha 1PI, and elastase inhibitory capacity. Samples were obtained during intervals of health and respiratory illness. BAL fluid from healthy alpha 1PI-deficient lung transplant recipients had minimal or unmeasurable free elastase activity, which was not different from that of control subjects. alpha 1PI concentrations in BAL fluid from alpha 1PI-deficient lung transplant recipients were reduced when compared with those of control subjects. Despite this observation, all but one alpha 1PI-deficient patient had the ability to inhibit exogenous elastase. During respiratory illness, however, three of seven alpha 1PI-deficient lung transplant recipients had measurable free elastase activity, which was inhibited ex vivo by addition of alpha 1PI. We conclude that alpha 1PI-deficient lung transplant recipients demonstrate free elastase activity in BAL fluid during severe lower respiratory tract inflammation, which is not present during health. Intravenous supplementation of alpha 1PI-deficient lung transplant recipients with exogenous alpha 1PI during respiratory tract inflammation may be indicated to inhibit elastase-mediated injury to the transplanted lung.