Increase in laminin expression in allergic airway remodelling and decrease by dexamethasone.

Lung expression of the extracellular matrix protein, laminin, and its receptor, laminin-1 receptor, were examined in a mouse model of asthma with airway remodelling. Ovalbumin (OVA) was administered to BALB/c mice, intraperitoneally on days 0 and 14, and intranasally periodically between days 14 and 75. The mice developed airway eosinophil and mononuclear inflammatory cell infiltration and fibrosis. On day 76, a marked increase in total laminin was seen in the airways of OVA-treated mice compared to controls by Western blot analysis. The increased laminin expression was detected immunocytochemically in the thickened subepithelial basement membrane and around airways and blood vessels. The OVA-treated mice showed increased expression of the alpha1, beta1, and gamma1 chains of the laminin-1 isoform in monocytes, macrophages and eosinophils infiltrating the airways. Laminin-1 receptor expression was increased in inflammatory and endothelial cells in the lungs of OVA-treated mice compared to controls. Treatment of OVA-sensitised/challenged mice with dexamethasone reduced airway expression of laminin and laminin-1 receptor in OVA-treated mice but not airway hyperresponsiveness to methacholine. Laminin deposition may be an important component of the airway remodelling observed in chronic allergic lung inflammation and is a process modulated by corticosteroids.

Expression of interleukin-5 and granulocyte-macrophage colony-stimulating factor in aspirin-sensitive and non-aspirin-sensitive asthmatic airways.

Increased numbers of eosinophils and mast cells in the bronchial mucosa are characteristic features in subjects with aspirin-sensitive asthma. Interleukin-5 (IL-5) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are involved in the activation, maturation, and perpetuation of survival of eosinophils. Immunohistochemical techniques were therefore used to study the expression of IL-5 and GM-CSF on frozen bronchial biopsies from 13 aspirin-sensitive asthmatic (ASA) and 8 non-ASA (NASA) subjects. Aspirin sensitivity was diagnosed by lysine-aspirin inhalation provocation. ASA airways demonstrated a significant 2-fold increase in the total number of submucosal inflammatory cells expressing IL-5 (p = 0.03) and approximate 4- and 2-fold increases in the numbers of mast cells expressing IL-5 and GM-CSF (p = 0.02 and p = 0.04, respectively). There was also a 4-fold increase in the number of eosinophils expressing IL-5 (p = 0.004). These results suggest a central role for the mast cell and eosinophil in regulation of the inflammatory cell infiltrate of ASA airways by secretion of the hemopoietic cytokines IL-5 and GM-CSF.

Blockade of CD49d (alpha4 integrin) on intrapulmonary but not circulating leukocytes inhibits airway inflammation and hyperresponsiveness in a mouse model of asthma.

Immunized mice after inhalation of specific antigen have the following characteristic features of human asthma: airway eosinophilia, mucus and Th2 cytokine release, and hyperresponsiveness to methacholine. A model of late-phase allergic pulmonary inflammation in ovalbumin-sensitized mice was used to address the role of the alpha4 integrin (CD49d) in mediating the airway inflammation and hyperresponsiveness. Local, intrapulmonary blockade of CD49d by intranasal administration of CD49d mAb inhibited all signs of lung inflammation, IL-4 and IL-5 release, and hyperresponsiveness to methacholine. In contrast, CD49d blockade on circulating leukocytes by intraperitoneal CD49d mAb treatment only prevented the airway eosinophilia. In this asthma model, a CD49d-positive intrapulmonary leukocyte distinct from the eosinophil is the key effector cell of allergen-induced pulmonary inflammation and hyperresponsiveness.

Enhanced expression of cyclo-oxygenase isoenzyme 2 (COX-2) in asthmatic airways and its cellular distribution in aspirin-sensitive asthma.

BACKGROUND: There are two isoforms of cyclo-oxygenase (COX), namely COX-1 and COX-2. COX-1 is constitutively expressed in most tissues and in blood platelets. The metabolites derived from COX-1 are probably involved in cellular housekeeping functions. COX-2 is expressed only following cellular activation by inflammatory stimuli and is thought to be involved in inflammation. METHODS: The expression of COX-1 and COX-2 isoenzymes has been studied in the bronchial mucosa of 10 normal and 18 asthmatic subjects, 11 of whom had aspirin-sensitive asthma (ASA) and seven had non-aspirin-sensitive asthma (NASA) RESULTS: There was a significant fourfold and 14-fold increase, respectively, in the epithelial and submucosal cellular expression of COX-2, but not of COX-1, in asthmatic patients. There was no significant difference in the total number of cells staining for either COX-1 or COX-2 between subjects with ASA and NASA, but the number and percentage of mast cells that expressed COX-2 was significantly increased sixfold and twofold, respectively, in individuals with ASA. There was a mean fourfold increase in the percentage of COX-2 expressing cells that were mast cells in subjects with ASA and the number of eosinophils expressing COX-2 was increased 2.5-fold in these subjects. CONCLUSION: COX-2-derived metabolites may play an essential part in the inflammatory processes present in asthmatic airways and development of drugs targeted at this isoenzyme may have therapeutic potential in the treatment of asthma. Mast cells and eosinophils may also have a central role in the pathology of aspirin-sensitive asthma.

Inflammatory cell populations in bronchial biopsies from aspirin-sensitive asthmatic subjects.

The inflammatory cell infiltrate in bronchial biopsies of 12 aspirin-sensitive asthmatic (ASA) subjects and eight non-aspirin-sensitive (non-ASA) control subjects have been compared. Biopsies were taken from a right middle or lower lobe segmental carina using fiberoptic bronchoscopy. The biopsies were snap-frozen in OCT, and sections 5 microns thick were doubled immunostained using a rabbit polyclonal antibody to the enzyme 5-lipoxygenase (5-LO) and with a monoclonal antibody to neutrophils (NP57), macrophages (EMB11), and total (BMK13) and activated eosinophils (EG2), mast cells (AA1), and T-lymphocytes (anti-CD3). There was no significant difference in the total numbers of cells staining for 5-LO between the two groups of subjects. As a percentage of total 5-LO cells, there were significantly more mast cells (12.9 +/- 3.8% versus 3.4 +/- 3.1%; p = 0.039) and total eosinophils (34.7 +/- 9.4% versus 11.1 +/- 3.8%; p = 0.044) and significantly fewer macrophages (23.3 +/- 6.1% versus 39.8% +/- 5.3; p = 0.041) in the bronchial biopsies from ASA subjects as compared with non-ASA patients. The numbers of neutrophils, T-lymphocytes, and activated eosinophils were similar for the two groups. The increased numbers of eosinophils and mast cells identified in the bronchial tissue from aspirin-sensitive asthmatic subjects may be the source of the enhanced cysteinyl leukotriene production observed in these subjects.

Effect of endobronchial aspirin challenge on inflammatory cells in bronchial biopsy samples from aspirin-sensitive asthmatic subjects.

BACKGROUND: The aspirin-induced bronchoconstriction in patients with aspirin-sensitive asthma is caused by cysteinyl leukotriene release. The cellular source of the leukotrienes is unknown. The inflammatory cell infiltrate in bronchial biopsy samples from seven aspirin-sensitive asthmatic (ASA) subjects and eight non-ASA subjects before and after local challenge with lysine aspirin was therefore examined. METHODS: Using flexible bronchoscopy, airway mucosal biopsy samples were taken and lysine aspirin solution was placed directly onto a carina of the contralateral lung. Twenty minutes later a second series of biopsy samples was taken from the site of the local endobronchial lysine aspirin challenge. The biopsy samples were double immunostained with a rabbit polyclonal antibody to the enzyme 5-lipoxygenase and monoclonal antibodies to mast cells (AA1), neutrophils (NP57), macrophages (EBM11), T lymphocytes (anti-CD3), and total (BMK13) and activated eosinophils (EG2). RESULTS: A decrease in both absolute mast cell numbers staining with mast cell tryptase (AA1) and the percentage of mast cells co-immunostaining with 5-lipoxygenase was seen in the ASA patients after lysine aspirin challenge compared with the non-ASA control group. There was also an increase in the numbers of activated eosinophils (EG2) in the ASA subjects compared with the non-ASA group. No changes were observed in the total numbers of macrophages (EBM11), neutrophils (NP57), total eosinophils (BMK13), and T lymphocytes (anti-CD3) after challenge with lysine aspirin. CONCLUSIONS: The decrease in numbers of mast cells staining for tryptase and the increase in activated eosinophils after endobronchial challenge with lysine aspirin may represent degranulation of these cell types, and may be an early event associated with aspirin-sensitive reactions in ASA subjects.

Effect of altitude on urinary leukotriene (LT) E4 excretion and airway responsiveness to histamine in children with atopic asthma.

Asthmatic subjects who are resident at altitude may experience a deterioration in lung function following a stay at sea level. To determine whether measurement of urinary leukotriene E4 (LTE4) reflects changes in asthma severity and airway responsiveness, 14 allergic asthmatic subjects resident at altitude (1560 m, Davos, Switzerland) were studied. Subjects were randomly divided into two groups. Measurements of baseline forced expiratory volume in one second (FEV1), the concentration of histamine producing a 20% decrease in FEV1, (PC20 FEV1), serum total immunoglobulin E (IgE), eosinophil count, and urinary LTE4 concentration were determined prior to and following a 2 week stay in The Netherlands (sea level) in eight subjects (4 males and 4 females, aged 14 +/- 0.5 yrs) (mean +/- SEM) and over a similar time period in six subjects (4 males and 2 females, aged 15 +/- 0.3 yrs) resident in Davos, Switzerland. There was no significant difference in total IgE and eosinophil count, and no significant correlation between urinary LTE4 and PC20FEV1 histamine, FEV1, total IgE, and eosinophil count. In subjects returning to Davos from The Netherlands there was a significant increase in urinary LTE4 from a baseline value of 16.9 pg.mg-1 creatinine (GM, range 0.3-101.7 pg.mg-1 creatinine) to 52.3 pg.mg-1 creatinine (GM, range 8.8-301.6 pg.mg-1 creatinine), a significant decrease in PC20FEV1 from 1.7 mg.ml-1 (GM, range 0.3-16.4 mg.ml-1) to 0.9 mg.ml-1 (GM, range 0.1-->32 mg.ml-1), and a significant fall in FEV1 from 3.0 +/- 0.3 to 2.8 +/- 0.3 l (mean +/- SEM).(ABSTRACT TRUNCATED AT 250 WORDS)

Increased urinary LTE4 excretion following inhalation of LTC4 and LTE4 in asthmatic subjects.

Urinary leukotriene E4 (LTE4) increases during exacerbations of asthma and following antigen challenge. We determined whether urinary LTE4 excretion reflects sulphidopeptide leukotrienes in the airways of asthmatic patients. Urinary LTE4 concentration was measured prior to and 1.5 and 3.5 h following inhalation of bronchoconstrictive doses of leukotriene C4 (LTC4) or LTE4 in eight asthmatic subjects. Increasing doses of agonist were inhaled until a 35% fall in specific airways conductance (sGaw) was achieved. There was no significant difference between the 53 +/- 3% (mean +/- SEM) fall in sGaw following inhalation of LTC4 (63.1 ng geometric mean, GM, range 5.8-527.5 ng) and the 43 +/- 4% fall in sGaw following inhalation of LTE4 7.94 ng/GM (range 132-3701 ng). The LTE4 excretion rate increased significantly from 2.95 (range 0.6-17.5) ng.h-1 to 4.67 (range 0.8-20) ng.h-1 at 1.5 h following LTC4 inhalation; and from 1.8 (range 0.07-6.7) ng.h-1 to 6.9 (range 2.9-27.3) ng.h-1 at 1.5 h following LTE4 inhalation; and had returned from baseline by 3.5 h. There was a correlation between the dose of LTC4 inhaled and LTE4 excreted in the urine (r = 0.82 and r = 0.72, respectively). The % recovery of LTE4 in the urine, of the total dose of inhaled LTC4 or LTE4 administered, was 6.9 +/- 4.1% and 0.8 +/- 0.3%, respectively. Thus, inhalation of bronchoconstricting doses of LTC4 or LTE4 alter urinary LTE4 excretion in a dose-dependent fashion. This indicates that urinary LTE4 can be used as a marker of sulphidopeptide leukotriene synthesis in the lungs of patients with asthma.

Airway responsiveness to leukotriene C4 (LTC4), leukotriene E4 (LTE4) and histamine in aspirin-sensitive asthmatic subjects.

We wanted to determine whether the airway response to inhaled leukotriene C4 (LTC4) is similar to inhaled leukotriene E4 (LTE4) in aspirin-sensitive asthma and, therefore, determined airway responsiveness to histamine, LTC4 and LTE4 in seven aspirin-sensitive subjects and 13 control asthmatic subjects, who were tolerant of aspirin. The concentration of inhaled lysine-aspirin which produced a 15% fall in forced expiratory volume in one second (FEV1) (PC15) was determined in aspirin-sensitive asthmatic subjects. The dose of histamine, LTC4 and LTE4 which produced a 35% fall in specific airways conductance (PD35sGaw) was determined by linear interpolation from the log dose response curve. There was no correlation between the PC15 for lysine-aspirin and the airway reactivity to inhaled LTC4 or LTE4. There was no difference in airway response to histamine and LTC4 between any of the groups of asthmatic subjects. There was a rank order of potency LTC4 > LTE4 > histamine in both groups, with LTC4 approximately 1,000 fold more potent than histamine in both groups. Aspirin-sensitive asthmatic subjects were significantly more responsive to LTE4 (p = 0.02) than aspirin-tolerant asthmatic subjects. The relative responsiveness of LTE4 to histamine (PD35 histamine/PD35 LTE4) was significantly greater in aspirin-sensitive asthmatic subjects compared to aspirin-tolerant asthmatic subjects (p = 0.05). There was no difference in relative responsiveness of LTC4 to histamine between aspirin-sensitive or aspirin-tolerant asthmatic subjects. We conclude that the airways of aspirin-sensitive asthmatic subjects demonstrate a selective hyperresponsiveness to LTE4, which is not observed for LTC4.

An analysis with sequence-specific oligonucleotide probes of the association between aspirin-induced asthma and antigens of the HLA system.

BACKGROUND: Previous work has suggested that in addition to environmental influences, there is a genetic predisposition for the development of both asthma and atopy. A subset of asthmatic subjects who are intolerant to aspirin has been identified. A previous study has suggested that there is an association between aspirin sensitivity and HLA-DQw2. METHODS: To further assess this association, we studied two populations of aspirin-sensitive subjects and aspirin-tolerant subjects with asthma. Genomic DNA was amplified by polymerase chain reaction and hybridized with radiolabeled oligonucleotide probes specific to the second exon of the DRB1, DQA1, DQB1, and DPB1 chains. RESULTS: Using polymerase chain reaction amplification of genomic DNA and sequence-specific oligonucleotide probes, we have been unable to confirm a significant association between aspirin sensitivity and HLA-DQw2. CONCLUSION: We have shown, however, that there is a significant decrease in the incidence of DPB1*0401 in both aspirin-tolerant and aspirin-intolerant subjects with asthma in both populations studied.

Effect of indomethacin on leukotriene4-induced histamine hyperresponsiveness in asthmatic subjects.

The effect of indomethacin on the capacity of LTE4 to enhance airway histamine responsiveness was evaluated in eight mild asthmatic subjects. Subjects attended the laboratory on three separate pairs of study days when inhalation challenges with methacholine or LTE4 were performed and the airway responses to histamine were measured 4 and 7 h later. An open pair of study days was followed by a pair of study days during ingestion of either placebo or indomethacin capsules. The dose of agonist that produced a 35% fall in specific airways conductance (PD35 SGaw) was obtained by linear interpolation from the logarithmic dose-response curve. Indomethacin treatment did not affect baseline SGaw or methacholine airway responsiveness. However, indomethacin significantly inhibited LTE4-induced histamine hyperresponsiveness. Maximum enhancement of histamine responsiveness by LTE4 on the open and placebo study days was 4.1 +/- 0.9- (mean +/- SEM) and 5.7 +/- 1.2-fold, respectively (p = 0.36). Maximal enhancement on the indomethacin day was 1.68 +/- 0.46, and this was significantly decreased compared with that on the placebo day (p = 0.02). This suggests that LTE4-induced enhanced responsiveness to histamine is mediated in part by cyclooxygenase pathway-derived products.

Urinary leukotriene E4 after lysine-aspirin inhalation in asthmatic subjects.

The FEV1 and urinary leukotriene E4 (LTE4) concentrations were determined in six aspirin-sensitive and six non-aspirin-sensitive asthmatic subjects before and after inhalation challenge with lysine-aspirin or placebo solution. Lysine-aspirin produced a mean fall in FEV1 of 26.7 +/- 4.9% (mean +/- SEM) in subjects with aspirin sensitivity and of 8.5 +/- 6.5% (mean +/- SEM) in non-aspirin-sensitive asthmatic subjects. The mean baseline urinary LTE4 concentration of 83 pg/mg creatinine (geometric mean [GM], range 15 to 326 pg/mg creatinine) in aspirin-sensitive subjects was significantly higher than the 33.8 pg/mg creatinine (GM, range 10 to 111 pg/mg creatinine) in non-aspirin-sensitive subjects (p = 0.02). In aspirin-sensitive subjects, inhalation challenge with lysine-aspirin produced a significant increase in urinary LTE4 concentration to 240 pg/mg creatinine (GM, range 60 to 1,113 pg/mg creatine), which was not observed after placebo challenge. There was no significant change in urinary LTE4 concentration after inhalation challenge with either lysine-aspirin or placebo solution in non-aspirin-sensitive asthmatic subjects. Thus, sulfidopeptide leukotrienes are released after inhalation of lysine-aspirin in aspirin-sensitive asthmatic patients.

Urinary leukotriene E4 in bronchial asthma.

Leukotriene E4 (LTE4) is excreted into the urine in a relatively constant proportion of 4-7% when either leukotriene C4 (LTC4) or LTE4 is intravenously infused, regardless of the magnitude of the infused dose. Measurement of LTE4 in urine is, therefore, a convenient and non-invasive method for assessing changes in the rate of total body sulphidopeptide leukotriene production. We assayed urinary LTE4 in 17 normal subjects, 31 subjects with asthma without aspirin sensitivity, and 10 aspirin-sensitive subjects. The relationship between urinary LTE4 and nonspecific bronchial hyperresponsiveness, as assessed by the provocative dose producing a 20% fall in forced expiratory volume in one second (PD20) to inhaled histamine, was examined in 19 non-aspirin-sensitive asthmatic subjects. The urinary LTE4 values were log-normally distributed. Urinary LTE4 was detected in 28 of the 31 non-aspirin-sensitive asthmatic subjects, and the geometric mean (95% confidence interval (CI) of 43 (32-57) pg.mg-1 creatinine was no different to that of 34 (25-48) pg.mg-1 creatinine measured in the normal subjects. The geometric mean of 101 (55-186) pg.mg-1 creatinine measured in the aspirin-sensitive asthmatics was significantly higher than that measured in the normal subjects (p less than 0.005) and in the asthmatic subjects who were non-aspirin-sensitive (p less than 0.002), but there was considerable overlap between the three groups. There was no relationship between urinary LTE4 and PD20, or between urinary LTE4 and baseline forced expiratory volume in one second (FEV1) (% predicted). Thus, measurement of LTE4 in a single sample of urine will not predict the extent of bronchial hyperresponsiveness or degree of airflow obstruction.

The effects of lipoxin A4 on airway responses in asthmatic subjects.

This study was performed to determine whether lipoxin A4 (LXA4) inhalation in asthmatic subjects has an effect on airways response. Eight subjects (six asthmatic, two normal) attended for bronchial inhalation challenge with LXA4. In three of these subjects (two asthmatics, one normal) blood pressure, pulse, and symptoms before and after challenge were recorded. Subsequently five male patients with mild asthma (22 to 34 yr of age) reattended for bronchial inhalation challenge with either leukotriene C4 (LTC4) or the combination of LTC4 and 1 x 10(-4) M LXA4. After inhalation of each dose of agonist SGaw and V25 were measured. Airway responsiveness was determined by the concentration of agonist in the nebulizer required to induce a 35% fall in SGaw (PC35). There was no effect of LXA4 inhalation on SGaw, V25, blood pressure, pulse, or symptoms. There was a significant shift of the SGaw and V25 dose-response curve to the right after inhalation challenge with LTC4 combined with 1 x 10(-4) M LXA4 as compared with that after inhalation challenge with LTC4 alone (p less than 0.01 and p less than 0.025, respectively). Thus, LXA4 may modulate LTC4-induced airway obstruction and may act as an endogenous sulfidopeptide leukotriene receptor antagonist.

Urinary leukotriene E4 levels after allergen and exercise challenge in bronchial asthma.

Urinary leukotriene E4 (LTE4) concentrations were measured in six asthmatic subjects after treadmill exercise, and in five asthmatic subjects after allergen challenge. Exercise and allergen challenge produced a 42 +/- 18% (mean +/- SD) and 22 +/- 8% fall in FEV1, respectively. The baseline concentration of urinary LTE4 in subjects challenged with exercise was 64 (27 to 150) pg/mg creatinine (geometric mean and 95% confidence interval), and in those challenged with allergen it was 36 (23 to 59) pg/mg creatinine. Urinary LTE4 concentrations did not change significantly in the 24 h after exercise. In contrast, there was a mean 4-fold increase in urinary LTE4 during the 3 h after allergen challenge.

The potent and selective sulfidopeptide leukotriene antagonist, SK&F 104353, inhibits aspirin-induced asthma.

We have determined the effect of prior inhalation of the LTD4 antagonist SK&F 104353 on the response to aspirin ingestion in six aspirin-sensitive asthmatic subjects (five women and one man 31 to 54 yr of age) in a randomized, double-blind, cross-over, placebo-controlled study. Pretreatment with inhaled SK&F 104353 (average nebulized dose, 893 micrograms) inhibited the response by a mean of 47% (p = 0.02). The inhibition was partial, ranging from 43 to 74% in five subjects. In the remaining subject, there was no effect of the drug on the asthmatic response. We conclude that the mechanism of aspirin-induced asthma is at least partially mediated by the leukotrienes in the majority of susceptible patients and that leukotriene antagonists may be useful in the treatment of aspirin-induced asthma.