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CONTENTS
ASTHMA AND AIRWAY BIOLOGY
ALLERGIC RHINITIS AND NASAL...
REFERENCES
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Asthma and Airway Biology (150)
Genetics (5)
Epidemiology (10)
Airway Inflammation (33)
Animal Models (8)
Induced Sputum (4)
Bronchial and Bronchoalveolar Specimens (7)
Blood (4)
Exhaled Nitric Oxide (4)
Other Exhaled Markers (4)
Review Articles (2)
Airway Hyperreactivity (27)
Animal Models: Antigen Challenge (10)
Animal Models: Other Challenges and Mediators (3)
Ex-vivo Studies (5)
Early and Late Asthmatic Responses (3)
Chemical and Antigen Challenge (3)
Hyperventilation- and Exercise-Induced Asthma (2)
Drugs (1)
Other Pathophysiologic Mechanisms in Asthma (31)
Tachykinins and Neural Activity (4)
Deep Inspiration (4)
Infection and Immunology (4)
Airway Narrowing (1)
Remodeling (17)
Review Article (1)
Treatment (30)
Beta-agonists (4)
Inhaled Glucocorticoids (6)
Glucocorticoids (1)
Theophylline (1)
Leukotriene Inhibitors (1)
Combination Regimens (4)
Immunotherapy (2)
Management Plans and Education (4)
New Agents (7)
Specific Clinical Scenarios (11)
Nocturnal Asthma (2)
Acute Severe and Fatal Asthma (2)
Severity and Chronicity (1)
Gastroesophageal Reflux (1)
Pregnancy (1)
Dyspnea (1)
Gas Exchange (1)
Quality of Life (1)
Psychopathology (1)
Occupational Asthma (3)
Laboratory Animal Workers (1)
Prevalence and Severity (2)
Allergic Rhinitis and Nasal Disorders (7)
Nasal Function (1)
Inflammation and Hyperreactivity (4)
Nasal Polyposis (2)
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ASTHMA AND AIRWAY BIOLOGY |
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CONTENTS
ASTHMA AND AIRWAY BIOLOGY
ALLERGIC RHINITIS AND NASAL...
REFERENCES
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Genetics
Studies that show an association between asthma-related phenotypes and alleles within a specific gene have generally
lacked statistical power. Hakonarson and coworkers (1) did a
case-control study of phenotypes associated with asthma and
24 candidate genes in 94 patients with atopic asthma and 94 control subjects without atopy or asthma. Phenotypic information in the cases consisted of a physician diagnosis of asthma,
skin reactivity to 12 aeroallergens, level of IgE, pulmonary
function, and response to methacholine challenge. The targeted genes that were sequenced included the cytokine gene
cluster on chromosome 5q31-33, the interleukin-4 receptor on
chromosome 16p12, the high affinity IgE receptor 
chain
(Fc
RI) on chromosome 11q13, and other genes encoding cytokines, chemokines, adhesion molecules, and various enzymes that are implicated in the pathogenesis of asthma.
Forty-two single nucleotide polymorphisms (SNPs) were genotyped in the 24 genes, with an average minor allele frequency
of 20.3% (asthma) and 20.7% (control). The allelic frequencies of the single nucleotide polymorphisms in the candidate
genes did not differ between patients and control subjects.
Linkage studies conducted in 269 patients with atopic asthma
and in 230 of their unaffected relatives uncovered no evidence
of linkage to markers associated with the 24 genes. The authors conclude that the study failed to produce evidence that
variations within 24 candidate genes for atopy and asthma
significantly influence the expression of phenotypes of atopic
asthma or contribute to the susceptibility for developing
atopic asthma. An editorial commentary by Weiss (2) accompanies this article.
In the promoter region of the gene encoding CD14 (a receptor for endotoxin), a transition of C-to-T at position 
159
is associated with atopic phenotypes in American children. To
determine whether or not the C allele of CD14/159 is associated with phenotypes of atopy and asthma, Koppelman and
coworkers (3) studied 159 probands with asthma and 158 spouses as controls from an adult Dutch population in which
linkage of IgE and bronchial hyper-responsiveness to chromosome 5q had been previously reported. Homozygotes for the
C allele of CD14/159 had a higher number of positive skin tests, a higher level of serum IgE in subjects with positive skin tests, and more self-reported allergic symptoms as compared
with subjects with the CT and TT alleles. The authors conclude that the C-to-T promoter polymorphism at position
159 in the CD14 gene results in the expression of a more severe atopic phenotype.
Two polymorphisms of the 2-adrenergic receptor gene at
codon 16 (arginine to glycine) and codon 27 (glutamine to
glutamate) are thought to influence airway responsiveness. To
assess the association of asthma with polymorphisms of the 2-adrenergic receptor gene and cigarette smoking, Wang and
coworkers (4) did a case-control study of 128 patients with
asthma and 136 control subjects living in rural China. Genotyping revealed a marginal interaction between smoking and
the 2AR-16 genotype. Compared with individuals who were
homozygous for the Gly-16 allele and who never smoked, the
homozygotes for the Arg-16 alleles who ever smoked had an
increased risk of asthma (odds ratio, 7.8). This association had
a dose-dependent relationship to the number of cigarettes
smoked. Asthma was not related with polymorphisms of the
2-adrenergic receptor gene at position 27. The authors conclude that homozygotes for the Arg-16 allele who smoke cigarettes are at increased risk of developing asthma.
The major macromolecular components of mucus consist of mucin proteins that are encoded by various MUC genes. Using the BLAST search and DNA-sequencing approach, Chen and coworkers (5) identified an expressed sequence tag (EST) clone in mice that shows great similarity to the 3' end of the human MUC5B gene. The clone was named 3pmmuc5b-1. A subsequent search of the mouse genome database with this sequence identified two overlapping genomic clones that contained the sequence for both 3pmmuc5b-1 and the mouse Muc5ac gene. The genomic order of the mouse Muc gene is 5'-Muc5ac-Muc5b-3'. The results suggest that the newly identified EST clone, 3pmmuc5b-1, is part of the 3' portion of the mouse Muc5b gene. In situ hybridization demonstrated that the putative mouse Muc5b message was expressed in a restricted manner under the tongue and in the region of the submucosal glands of the trachea. In mice with ovalbumin-induced asthma, gene expression was greatly enhanced in airway surface epithelium and in submucosal glands. The authors conclude that the newly cloned mouse Muc5b gene could be used as a marker for studying aberrant gene expression in mouse models of airway diseases.
Epidemiology
To determine whether or not a diagnosis of asthma is associated with weight gain or physical activity, Beckett and coworkers (6) followed 4,547 young adults, 18 to 30 years of age at study entry, for 10 years. At study entry, a diagnosis of asthma was associated with center of the study, race, sex, low level of education, and active smoking, but not with exposure to environmental tobacco smoke. At baseline, asthma showed little association with body mass index. On follow-up, a new diagnosis of asthma showed a J-shaped pattern of association with body mass index among women, but not among men. (A J-shaped pattern occurs when a risk for the lowest quintile [lowest change in weight over 10 years] is slightly higher than for the second quintile, and when the risk for the three highest quintiles is more elevated.) A lower level of physical activity did not explain the association between the incidence of asthma and the gain in weight. The authors conclude that a gain in weight is associated with a new diagnosis of asthma in young women, and that a lower level of physical activity does not explain the association.
To determine the relationship between body weight and asthma, Celedon and coworkers (7) did a cross-sectional study of 7,109 adults from families of subjects with asthma in Anqing (a rural province in China). Criteria for asthma were a physician diagnosis of asthma plus airway responsiveness to methacholine (at 25 mg per ml or less) plus two or more respiratory symptoms or attacks of asthma. Criteria for symptomatic airway hyperresponsiveness were airway responsiveness to methacholine (at 8 mg per ml or less) plus two or more respiratory symptoms or attacks of asthma. After adjusting for intensity of cigarette smoking and other factors, multivariate analysis revealed that both being underweight and being overweight was associated with asthma in women, and that being underweight was associated with asthma in men. Compared with a body mass index (in kg per m2) of 21, the odds of symptomatic airway hyperresponsiveness was 2.5 times higher in men and 2 times higher in women for a body mass index of 16, and it was 2.3 times higher in both men and women for a body mass index of 30. The authors conclude that both being underweight and overweight is associated with an increased risk of asthma among adults in families of subjects with asthma that live in rural China.
To determine whether or not growing up on a farm protects against the development of allergy, Leynaert and coworkers (8) analyzed data from 6,251 randomly selected participants (aged 20 to 44 years) in the European Community Respiratory Health Survey. After adjusting for potential confounders, living on a farm during childhood was associated with a decrease in the risk of atopic sensitization as an adult (odds ratio, 0.76). Compared with other individuals, individuals growing up on a farm were less frequently sensitized to cats (odds ratio, 0.63) and Timothy grass (odds ratio, 0.68), and had a lower risk of nasal symptoms in the pollen season (odds ratio, 0.80). The authors conclude that growing up on a farm decreases the risk of atopy and hay fever in adulthood, suggesting that exposure to environmental factors in childhood may have a lifelong protective effect against the development of allergy.
In the developing countries, the move to cities is associated with a switch from biomass fuels (wood, charcoal, animal dung) to modern fuels (kerosene, gas, electricity). To assess the influence of exposure to modern fuels on the risk of allergy, Venn and coworkers (9) studied a random sample of individuals who live in Jimma, Ethiopia. Questionnaire information was collected from 9,844 adults and children, and allergy skin tests were done on 2,372 of the individuals. The use of any modern fuel was reported by 959 individuals (10%). Compared with the sole use of a biomass fuel, the use of a modern fuel was associated with an increased risk of allergic sensitization (odds ratio, 1.8), wheeze (odds ratio, 1.6), rhinitis (odds ratio, 2.1), and eczema (odds ratio, 2.8). The authors conclude that domestic combustion of modern fuels increases the risk of allergic sensitization and symptoms.
To determine the relationship between asthma or rhinitis and the reactivity of skin tests to aeroallergies in a rural area of mainland China, Celedon and colleagues (10) studied 10,009 members of 2,544 families selected on the basis that at least two siblings had physician-diagnosed asthma. Although 47% of the subjects were sensitized to at least one aeroallergen, only 3.5% reported symptoms of allergic rhinitis. Sensitization to dust mite, a perennial aeroallergen, was a predictor of asthma (odds ratio, 1.3) and rhinitis (odds ratio, 1.3), and sensitization to mold was a predictor of asthma (odds ratio, 1.6). Sensitization to silk, a seasonal aeroallergen, was the strongest predictor of rhinitis (odds ratio, 1.5). The authors conclude that sensitization to perennial aeroallergens is predictive of asthma among subjects in rural China who have a family history of asthma, and that allergic rhinitis is far less common in rural China as compared with the industrial world.
To determine whether or not indoor levels of common allergens influence sensitization and clinical activity of asthma, Dharmage and coworkers (11) studied 485 individuals. Fungal levels in air samples from bedrooms were associated with increased bronchial hyperreactivity. The level of ergosterol, an estimate of fungal biomass, in floor dust was a risk factor for sensitization to fungi and for having wheezed in the preceding year. A high level of Fel d 1 (cat allergen) in floor dust was associated with an increased risk of being sensitized to cats, and a high level of Fel d 1 in beds was associated with current asthma. None of the studied outcomes was related to the levels of Der p1, the house dust mite allergen, possibly because almost every participant was exposed to supranormal levels of this allergen. The authors conclude that exposure to fungal and cat allergens, but not to house dust mite allergen, has a significant influence on sensitization and the clinical activity of asthma in young adults.
To measure the incidence of asthma in Spain, Basagana and coworkers (12) did a follow-up study in 1998 and 1999 of 1,640 individuals who had previously participated in the European Community Respiratory Health Survey of 1991 to 1993. The subjects had been 20 to 44 years of age during the survey. Incident cases were defined as those who were free of asthma in 1991 to 1993 and who gave a positive answer to the question "have you ever had asthma" in 1998 to 1999. The incidence of asthma was 5.53 per 1,000 person years: 6.88 in women and 4.04 in men. The incidence was highest in the subjects who at the time of the baseline survey had bronchial hyperresponsiveness (incidence rate ratio, 3.85), positive IgE against Timothy grass (incidence rate ratio, 3.16), and who were women (incidence rate ratio, 1.80). No association was seen with total IgE, atopy, smoking, occupational exposure, or maternal asthma. The authors conclude that bronchial hyperresponsiveness and IgE reactivity to grass are the main determinants of new asthma in subjects who had not reported having asthma when assessed six years earlier.
Dietary antioxidants may protect against the development of asthma, but the evidence is weak. To investigate this relationship, Shaheen and coworkers (13) did a population-based study of 607 cases of asthma and 864 control subjects. After controlling for confounding variables, asthma was negatively associated with apple consumption (odds ratio per increase in frequency group, 0.89) and with the intake of selenium (odds ratio per quintile increase, 0.84). The severity of asthma was negatively associated with the consumption of red wine. The authors conclude that the associations between asthma with apples and red wine suggest that flavonoids and other dietary antioxidants may protect against asthma.
The association between cleanliness and the increase in asthma is debated by von Mutius (14) and Platts-Mills and colleagues (15), with rebuttals from each (16, 17).
Airway Inflammation
Animal models The eosinophilic inflammatory response in asthma is associated with protein nitration, which can be studied using immunostaining for 3-nitrotyrosine. In murine models of allergic asthma, Duguet and coworkers (18) investigated the relative contribution of eosinophil peroxidase and inducible nitric oxide synthase to protein nitration. Three strains of mice were sensitized and challenged intranasally with ovalbumin. Staining for 3-nitrotyrosine in eosinophils around the airways was much less in New Zealand White mice, which have a spontaneous deficiency of eosinophil peroxidase, as compared with A/J and C57BL/6J mice. Mice with a targeted deletion of inducible nitric oxide synthase and also wild-type control mice had similar staining for 3-nitrotyrosine. The authors conclude that protein nitration, after allergen challenge in a murine model of asthma, is dependent on eosinophil peroxidase in the eosinophils and it is not dependent on increased production of nitric oxide. An editorial commentary by Cross and colleagues (19) accompanies this article.
Horses with heaves have reversible airway obstruction, bronchial hyperresponsiveness, and lower airway inflammation. To determine whether or not affected horses have a cytokine profile similar to that seen in patients with asthma, Lavoie and coworkers (20) did bronchoalveolar lavages in seven horses with heaves and in five control horses. Compared with the control horses, the horses with heaves had increased expression of messenger RNA encoding interleukin-4 and interleukin-5, and decreased expression of interferon-
. The authors conclude that inflammatory cells in the lungs from horses with
heaves display a Th2-type of cytokine profile, suggesting that
heaves is an allergic condition with similarity to human asthma.
, interleukin-6, and
macrophage inflammatory protein-2. Instilling both monocyte chemotactic protein-1 and endotoxin into the trachea produced a 22-fold increase in neutrophils (peaking at 12 hours),
an eight-fold increase in alveolar monocytes (peaking at 48 hours), and large increases in the proinflammatory cytokines
and lung vascular leakage. When the same combination was
administered via the peritoneum, the synergistic response did
not occur. Blocking neutrophil recruitment with anti-CD 18 did not affect the cytokine response to the combination. The
authors conclude that the combination of monocyte chemotactic protein-1 and endotoxin in the alveolar compartment
have a synergistic effect, producing an early inflammatory response with increased cytokine synthesis and neutrophil recruitment, and a late phase of enhanced monocyte traffic and
expansion of the alveolar macrophage pool.
CD4+ CD25+ T cells are immunoregulatory cells that prevent autoimmune diseases mediated by CD4+ T cells. To determine the role of CD4+ CD25+ T cells in allergic inflammation of
the airways, Suto and coworkers (23) studied antigen-induced
recruitment of eosinophils in the airways of BALB/cRag-2
/
mice. Mice transferred with CD4+ CD25+ T cell-depleted
splenocytes from ovalbumin-specific TCR transgenic mice recruited fewer eosinophils in response to antigen than mice transferred with unfractionated T cells. Depletion of CD4+
CD25+ T cells increased the recruitment of neutrophils and T
cells in the airways in response to antigen, caused depression
of interleukin-4 and interleukin-5 in the airways in response to
antigen, prevented the differentiation of type 2 (Th2) helper T
cells in vitro in response to antigen, and increased the differentiation of type 1 (Th1) helper T cells. The authors conclude
that CD4+ CD25+ T cells modulate the differentiation of
helper T cells toward the Th2 type and thus upregulate Th2
cell-mediated allergic inflammation in the airways.
Certain serine proteases can send signals to cells by cleaving proteinase-activated receptors (PARs). Trypsin and tryptase, found in mast cells, activate proteinase-activated receptor 2, and thrombin activates proteinase-activated receptors 1, 3, and 4. To determine the localization and function of proteinase-activated receptor 2, Schmidlin and coworkers (24) studied cultured smooth-muscle cells from human airways. The receptor was detected in the smooth muscle, epithelium, glands,
and endothelium of human bronchi. Agonists of the proteinase-activated receptor 2-trypsin, tryptase, and an activating
peptide (SLIGKV-NH2) stimulated the mobilization of calcium in the smooth-muscle cells. These agonists strongly desensitized the responses to a second challenge with trypsin and
SLIGKV-NH2, but not to thrombin, indicating that the agonists activate a receptor distinct from the thrombin receptors.
Contraction was increased by removing the epithelium and it
was diminished by indomethacin. The authors conclude that
proteinase-activated receptor 2 is expressed in human bronchial smooth muscle, where its activation mobilizes intracellular calcium and induces contraction.
Phosphatidylinositol 3-kinase is involved in the transduction
pathways of mediators produced by eosinophils. In a series of studies, Ezeamuzie and coworkers (25) investigated the effect of
worthmannin, an inhibitor of phosphatidylinositol 3-kinase. In in
vitro studies, worthmannin inhibited the release of superoxide and eosinophil peroxidase by eosinophils in response to stimulation with complement C5a. Both a challenge with aerosolized allergen in sensitized guinea pigs and the intravenous injection of
Sephadex beads in normal guinea pigs caused increased numbers
of eosinophils, increased eosinophil peroxidase, and airway hyperresponsiveness (in response to intravenous acetylcholine and
histamine). In the allergic model, intranasal pretreatment with
worthmannin had no effect on the eosinophilia, but it decreased
eosinophil peroxidase and abolished airway hyperresponsiveness to histamine (but not to acetylcholine). In the Sephadex
model, worthmannin inhibited the eosinophilia, the increase in
eosinophil peroxidase, and the airway hyperresponsiveness. The
authors conclude that worthmannin is a potent inhibitor of human eosinophil degranulation, and that intranasal administration of worthmannin prevents airway hyperresponsiveness in
guinea pigs through inhibition of eosinophil degranulation.
Induced sputum
To assess the safety and efficacy of sputum induction in patients with severe asthma, ten Brinke and
coworkers (26) studied 93 patients with severe asthma. The patients were symptomatic despite using a regular inhaled glucocorticoid and a long-acting 2-agonist for at least a year, and
had received at least one course of an oral glucocorticoid in the
preceding year. Patients with an FEV1 of less than 50% of predicted were excluded. A strict protocol for inhaling 0.9, 3.0, and 4.5% saline generated an adequate sputum sample in 74%
of the patients. A greater than 15% fall in FEV1 accompanied
the induction of sputum in 22% of the patients, and it occurred
despite pretreatment with albuterol. The decrease in FEV1 was
associated with an increase in the use of 2-agonists for rescue
in the preceding two days (rs = 0.51), a lower post-bronchodilator FEV1 (rs = 0.31), and a lower PC20 on methacholine provocation (rs = 0.52). The risk of excessive bronchoconstriction
was increased 10.2-fold in the patients who recently used 2-
agonists as rescue medication. The authors conclude that sputum induction can be successful and safe in patients with severe
asthma provided that a strict protocol is followed, and that the
patients at greatest risk of excessive bronchoconstriction are
those who used 2-agonists for rescue in the preceding days.
Bronchial and bronchoalveolar specimens
The cysteinyl-leukotrienes generated by 5-lipoxygenase in mast cells and
eosinophils, and also the prostanoid products generated by the
cyclooxygenase pathway in mast cells and Th2-lymphocytes, cause bronchoconstriction, leukocyte recruitment, and bronchial hyperresponsiveness in asthma. To characterize the cellular expression of the enzymes of these two pathways in the
bronchial mucosa, Seymour and coworkers (30) did bronchial
biopsies in 12 patients with atopic asthma both before and during seasonal exposure to birch pollen. During the pollen season, symptom scores for asthma increased by 3.8-fold, accompanied by a decrease in PEF and an increase in bronchial
responsiveness to methacholine. Bronchial biopsies during the
pollen season revealed two-fold increases in cells staining for
5-lipooxygenase, 5-lipooxygenase-activating protein, and leukotriene A4 hydrolase, and a fourfold increase in leukotriene
C4 synthase (the terminal enzyme in the synthesis of cysteinyl-leukotriene). The increase in leukotriene C4 activity was accompanied by a six-fold increase in the proportion of eosinophils staining for the enzyme. Macrophages were also increased,
but mast cells and subsets of T lymphocytes did not change.
Staining for enzymes of the cyclooxygenase pathway did not
change during the pollen season. Morning PEF was correlated
with the counts of cells staining for 5-lipooxygenase in biopsies
both before (r = 0.59) and during (r = 0.65) the pollen season. The authors conclude that the bronchial mucosa of patients with asthma shows increased activity of enzymes of the
5-lipoxygenase pathway, but not of the cyclooxygenase pathway, during allergen exposure, and that the increased activity is
mainly seen in airway eosinophils and macrophages.
0.37). The thickness of
the reticular basement membrane was 40% greater in the subjects in remission, as compared with the healthy subjects. The
authors conclude that young adults in remission of atopic
asthma display ongoing airway inflammation and remodeling.
Peroxisome proliferator-activated receptors belong to the
nuclear hormone receptor superfamily of ligand-activated transcription factors. In 34 patients with asthma, Benayoun and
coworkers (32) found that expression of peroxisome proliferator-activated receptor was increased in the bronchial submucosa, airway epithelium, and smooth muscle. The increased
expression was associated with enhanced proliferation and
apoptosis of airway epithelial and submucosal cells, and it was
associated with evidence of remodeling, including the thickness of the subepithelial membrane and deposition of collagen.
Expression of the receptor in the epithelium was positively
correlated with the thickness of the subepithelial membrane
(r = 0.67), and it was negatively correlated with FEV1 (r = 0.46). Glucocorticoids, inhaled or oral, downregulated cell proliferation, thickness of the subepithelial membrane, collagen deposition, and the expression of the receptor in all
compartments, and they increased the numbers of apoptotic
cells in the epithelium and submucosa. The authors conclude
that peroxisome proliferator-activated receptor is a marker
of airway inflammation and remodeling in asthma, and that it
is a target for glucocorticoid therapy.
Eotaxin is a chemokine of the CC class that selectively attracts eosinophils by activating the CCR3 receptors. To determine the role of eotaxin in asthma, Lilly and coworkers (33)
did segmental allergen challenges in six patients with atopic
asthma and in five healthy nonatopic subjects. Before the allergen challenge, the levels of eotaxin in the bronchoalveolar
fluid of the patients were 67% higher than in the control subjects. The challenge produced a 2.4-fold increase in eotaxin in
the bronchoalveolar fluid of the patients, accompanied by
macrophages and eosinophils that were immunopositive for
eotaxin, and increases in eotaxin expression in airway epithelial and endothelial cells. The levels of eotaxin were related to
the recovery of eosinophils in bronchoalveolar fluid (r2 = 0.88). The authors conclude that segmental allergen challenge in patients with asthma induces rapid epithelial production of airway and alveolar eotaxin in amounts sufficient to explain
the recruitment of eosinophils to those sites.
To determine whether or not allergic inflammation of the
airways increases the local production of complement factors
C3a and C5a, Krug and coworkers (34) did segmental bronchial challenges in 14 patients with mild asthma and in 9 healthy subjects. Twenty-four hours after the challenge, the
patients with asthma developed an 8.5-fold increase in C3a
and a 13.5-fold increase in C5a in bronchoalveolar fluid. No
change occurred in the control subjects. The number of eosinophils was correlated with the concentrations of both C3a (r = 0.90) and C5a (r = 0.88). The number of neutrophils was also
correlated with both C3a (r = 0.66) and C5a (r = 0.71). The
authors conclude that the anaphylatoxins C3a and C5a contribute to the pathogenesis of asthma.
To determine the interrelationships between goblet cells,
mucin, and airway function, Ordonez and coworkers (35) did
endobronchial biopsies in 13 patients with asthma and in 11 healthy subjects. Stored mucin was three times higher in goblet
cells of the patients with asthma as compared with the healthy
subjects. The number of goblet cells was 2.2 times higher in the
patients with asthma, although the size of the cells did not differ
from the healthy subjects. Patients with an FEV1 of less than
80% of predicted secreted 60% less mucin than patients who
had a higher FEV1. The secreted mucin was inversely related to
stored mucin (r = 0.78). The authors conclude that patients
with even mild asthma display goblet cell hyperplasia and increased storage of mucin in the epithelium, but that secretion of
mucin is increased only in patients with moderate asthma.
To determine whether or not expression of epidermal
growth factor receptor is related to the synthesis of mucin in
human bronchi, Takeyama and coworkers (36) did bronchial
biopsies in 12 patients with asthma and in 11 healthy subjects.
Messenger RNA for epidermal growth factor receptor was expressed in the airway epithelium, mainly in goblet cells, basal
cells, or both. Healthy airways showed little expression. Expression of messenger RNA for MUC5AC (a marker of goblet cell mucin) was increased in the airways of patients with
asthma, and expression was proportional to that of epidermal
growth factor receptor (r = 0.73). The authors conclude that
expression of epidermal growth factor receptor and MUC5AC (a marker of goblet cell mucin) is upregulated in the epithelium of the asthmatic airway and that their colocalization in
goblet cells may explain the mechanism for the synthesis of
mucin in airway epithelium.
Blood
Interleukin-4 is central to the maturation of type 2 (Th2) helper T cells and IgE class switching. The splice variant
of interleukin-4, IL-4
2, may be a functional antagonist of interleukin-4 that competes for receptor binding. Seah and coworkers (37) measured messenger RNA copy numbers of interleukin-4 and its splice variant in the peripheral blood monocytes of
9 patients who had chronic atopic asthma and high serum IgE
titers, in 18 patients with tuberculosis (a disease involving a significant increase in type-2 cytokine expression, although type-1
cytokines constitute the dominant response), and in 18 healthy
subjects. The median number of messenger RNA copy numbers of interleukin-4 in the patients with asthma was 2.8 logs
higher as compared with the patients with tuberculosis, and 4.5 logs higher as compared with the healthy subjects. The expression of the splice variant of interleukin-4, IL-42, in cells from
patients with asthma was similar to that seen in the cells from
patients with tuberculosis. The ratio of interleukin-4 to its splice
variant was 500 times higher in the patients with asthma than in
patients with tuberculosis or the healthy subjects. The authors
conclude that the low level of expression of the splice variant,
IL-42, relative to the expression of interleukin-4 may be a factor in the pathogenesis of asthma.
or CD11b expression. The authors conclude that systemically administered interleukin-5 increases the number of CD34+ eosinophil progenitor cells and the number of mature eosinophils expressing
the chemokine receptor CCR3 in patients with asthma.
Cytokines produced by type 2 (Th2) helper T cells are essential in the initiation and prolongation of the asthmatic response. The ST2 gene is preferentially expressed on Th2 cells
and appears to play an essential role in the development of
Th2 responses in patients with asthma. Using an ELISA system, Oshikawa and coworkers (39) found that the level of the
soluble form of human ST2 protein was 19% higher in the serum of 56 patients with atopic asthma than in that of 200 healthy subjects. In 30 patients experiencing an acute exacerbation of asthma, the serum level of ST2 protein was increased
4.8-fold. During the exacerbation, the level of ST2 protein was
correlated with percentage of predicted PEF (r = 0.63) and
with PCO2 (r = 0.52). The authors conclude that increase in
soluble human ST2 protein in the sera of patients with an
acute exacerbation of asthma may reflect the severity of Th2-dominant allergic inflammation.
When stimulated by antigen-presenting cells, naïve T cells differentiate into type 1 (Th1) helper T cells, which promote cellular
immune responses, and type 2 (Th2) helper T cells, which promote allergic responses. The CXC chemokine receptor (CXCR) 3 is expressed preferentially on Th1 cells, and the CC chemokine receptor (CCR) 4 is selectively expressed on Th2 cells. To determine whether or not oral prednisone would alter the balance between these two sets of T cells in the peripheral blood, Kurashima and coworkers (40) studied 28 patients with asthma and 13 healthy subjects before and after two weeks of oral prednisone
(20 mg per day) using a double-blind parallel design. The patients
developed a 31% decrease in CCR4+ T cells (expressed as a
fraction of CD4+/CD45RO+ memory T cells), no change in
CXCR3+ T cells, and a 55% increase in the ratio of CXCR3+ to
CCR4+ T cells. The healthy subjects showed no change in the
percentage of CCR4+ cells after glucocorticoid therapy. The authors conclude that a decrease in the proportion of CCR4+ T
cells in relation to CXCR3+ T cells may contribute to the beneficial action of glucocorticoids in asthma.
Exhaled nitric oxide
To assess the usefulness of exhaled
nitric oxide in detecting and predicting loss of control in
asthma, Jones and coworkers (41) withdrew inhaled glucocorticoid therapy from 78 patients with mild-to-moderate asthma.
Over the subsequent six weeks, 60 patients (78%) experienced
a deterioration in the control of their asthma, with a median
time to loss of control of 17 days. Exhaled nitric oxide was repeated every week, and the change in its concentration was
correlated with symptom score (r = 0.45), sputum eosinophils (r = 0.44), percent predicted FEV1 (r = 0.35), and the dose
of saline causing a 15% decrease in FEV1 (PD15; r = 0.45).
Both single and repeated measurements of exhaled nitric oxide had positive predictive values of 80 to 90% for predicting
loss of control. Sputum eosinophils and PD15 for hypertonic
saline had equivalent positive predictive values. The authors
conclude that exhaled nitric oxide is as powerful as the analysis of induced sputum or an airway challenge with hypertonic
saline in predicting the loss of asthma control, but is easier to
perform. An editorial commentary by Kharitonov and Barnes
(42) accompanies this article.
0.55). Glucocorticoid therapy decreased the abnormal patterns in both patient groups. The authors conclude that monitoring the bronchial flux and alveolar fractions of nitric oxide
may help in assessing disease activity at the bronchial level, alveolar level, or both.
Weicker and colleagues (44) examined the feasibility of
measuring exhaled nitric oxide in spontaneously breathing
mice. After placing a mouse in a Plexiglass chamber and allowing it to acclimatize, exhaled gas was collected. The mean concentration of exhaled nitric oxide was 10 ppb and the maximal
day-to-day variation was 2 ppb in an individual animal. Administration of NG-nitro-L-arginine methyl ester (L-NAME), a nonselective inhibitor of nitric oxide synthase, produced a 51% decrease in exhaled nitric oxide. Acute lung injury secondary to
intraperitoneal administration of lipopolysaccharide caused a
30% increase in exhaled nitric oxide. The authors conclude
that their methodology permits noninvasive measurement of
exhaled nitric oxide in a spontaneously breathing mouse.
Other exhaled markers To investigate the relationship between exhaled carbon monoxide and airway inflammation, Khatri and coworkers (45) exposed eight patients with atopic asthma to a whole lung allergen challenge. At baseline, exhaled carbon monoxide was equivalent in the patients and in the control subjects: 1.9 versus 1.8 ppm. After the allergen challenge, the patients developed an immediate decrease in exhaled carbon monoxide (to 1.4 ppm) and it returned to baseline after 1 hour. At baseline, exhaled nitric oxide was higher in the patients as compared with the control subjects: 15 versus 7.3 ppb. All but one patient developed an increase in exhaled nitric oxide at three hours after allergen challenge. The authors conclude that allergen challenge produces an immediate decrease in exhaled carbon monoxide in patients with asthma and that the levels return to baseline during the late asthmatic response.
Nitrosothiols, formed by the interaction of nitric oxide with glutathione, help stabilize nitric oxide in a form that is not cytotoxic. Corradi and coworkers (46) measured nitrosothiols in exhaled breath condensates of patients with inflammatory airway diseases. The levels of nitrosothiols were 0.11 µM in 10 healthy subjects, 0.08 µM in nine patients with mild asthma, 0.81 µM in eight patients with severe asthma, 0.35 µM in 10 patients with cystic fibrosis, 0.24 µM in seven patients with COPD, and 0.46 µM in seven normal smokers. In current smokers, nitrosothiol was related to the packs of cigarettes smoked (r = 0.80). The level of nitrite in exhaled air was elevated in the patients with severe asthma, patients with cystic fibrosis and patients with COPD, but not in the patients with mild asthma or the smokers-suggesting that it is less sensitive than exhaled nitrosothiols. The authors conclude that nitrosothiols are increased in exhaled breath condensates of patients with inflammatory airway diseases. In a state-of-the-art review article, Kharitonov and Barnes (47) provide a detailed and comprehensive discussion of exhaled markers in pulmonary disease. In a pulmonary perspective, Mutlu and colleagues (48) discuss the collection and the analysis of exhaled breath condensates.Review articles In a state-of-the-art review article, D'Ambrosio and colleagues (49) discuss the role of cytokines and their receptors in guiding the recruitment of T lymphocytes in lung inflammation.
In a pulmonary perspective, Salvi and colleagues (50) discuss the role of a polarized response of T cells toward a helper type 2 phenotype in the causation of asthma.Airway Hyperreactivity
Animal models: antigen challenge To determine the relative roles of isoforms of nitric oxide synthase in contributing to airway hyperresponsiveness, Samb and coworkers (51) studied lung homogenates and tracheal smooth muscle from guinea pigs immunized and repeatedly challenged with ovalbumin. Six hours after the preceding challenge, protein expression of nitric oxide synthase 1 (the neural isoform) was decreased in lung homogenates, and returned to baseline at 24 hours. Nitric oxide synthase 3 was not modified, and nitric oxide synthase 2 was undetectable. The decreased expression of nitric oxide synthase 1 was associated with a decrease in the conversion of L-[3H]arginine to L-[3H]citrulline and a decrease in the concentrations of nitrate and nitrite in the lung. The decreased expression of nitric oxide synthase 1 was accompanied by a decrease in exhaled nitric oxide and by the development of airway hyperresponsiveness to histamine. The authors conclude that ovalbumin stimulation in guinea pigs induces a transient decrease in the expression and activity of nitric oxide synthase 1, which probably participates in airway hyperresponsiveness.
To determine the role of eosinophils in nitric oxide mediated injury, Iijima and coworkers (52) studied mice sensitized with ovalbumin. A challenge with ovalbumin caused eosinophils to increase from 0 to 60% of cells in bronchoalveolar fluid, accompanied by a 40% increase in nitric oxide metabolites. Inducible nitric oxide synthase was expressed in airway epithelial and inflammatory cells, and 3-nitrotyrosine was found in peribronchial inflammatory cells and at the epithelial surface. The nitric oxide metabolites and 3-nitrotyrosine were reduced by pretreatment with a specific inhibitor of nitric oxide synthase, 1400W, and by a nonselective inhibitor, Nw- nitro-L-arginine methyl ester. The specific inhibitor produced a 62% decrease in the number of eosinophils, suggesting that the production of nitric oxide contributed to the eosinophil recruitment. Pretreatment with an antibody to interleukin-5 produced a 90% decrease in the eosinophilia that follows ovalbumin challenge, decreased the nitric oxide metabolites to baseline, and produced a 74% decrease in cells positive for 3-nitrotyrosine. The authors conclude that nitric oxide and eosinophilia are closely coupled and that eosinophils are important in protein nitration. Early growth-response factor 1 is a transcription factor that plays a regulatory role in the expression of many genes. To determine the role of this factor in airway inflammation and reactivity, Silverman and coworkers (53) studied wild-type mice and knockout mice lacking the transcription factor. In response to ovalbumin sensitization and airway challenge, the knockout mice had lower levels of messenger RNA and protein of tumor necrosis factor- in the lungs and mast cells. At
baseline and after allergen challenge, the knockout mice had
elevated levels of IgE, and their airways were less responsive
to methacholine. The authors conclude that early growth-
response factor 1 modulates the expression of tumor necrosis
factor-, the production of IgE, and responsiveness of the airways of mice.
Two 4 integrins have been characterized. One is 41 (also
known as very late antigen-4), which is an adhesion receptor
that interacts with vascular cell adhesion molecule-1. The second is 47, which is expressed on lymph node T and B cells,
natural killer cells, and eosinophils. Ramos-Barbon and coworkers (54) determined whether or not the blockade of the
late airway response by 4 integrins is mediated by T cell activation. CD4+ T cells were taken from sensitized rats and given
to non-sensitized rats, and the latter developed a late reaction when subsequently challenged with ovalbumin. Rats
treated with TA-2, an antibody to 4-integrin, developed
fewer cells expressing messenger RNA for interleukin-5 and
fewer eosinophils in bronchoalveolar fluid as compared with a
placebo group. Counts of total cells, macrophages, neutrophils
and lymphocytes were unaffected. Expression of interferon- was downregulated in rats receiving an allergen challenge,
consistent with activation of type 2 (Th2) T cells and reciprocal inhibition of Th1 cytokines. The authors conclude that attenuation of the late response and eosinophilia by blockade of
the 4-integrin adhesion receptors may involve interference
with CD4+ cell activation and interleukin-5 expression.
To better understand the mechanism of action of rolipram,
a specific inhibitor of phosphodiesterase 4, Kanehiro and coworkers (55) studied airway inflammation and function in a
model of secondary allergen challenge. Mice were sensitized and
challenged with ovalbumin (primary challenge). On re-exposure
to ovalbumin after six weeks (secondary challenge), the mice
had increased numbers of inflammatory cells and interleukin-4
and interleukin-5 in bronchoalveolar fluid. Rolipram caused a
dose-dependent decrease in eosinophil, lymphocyte, and neutrophil accumulation, reduced the levels of interleukin-4 and
interleukin-5, and prevented the changes in resistance and
compliance that occur on challenge with methacholine. In contrast with rolipram, antibodies to very late activating antigen-4
and to interleukin-5 prevented only the increases in resistance,
eosinophil numbers, and interleukin-5. Goblet cell hyperplasia was suppressed by rolipram, but not by the other two therapies. The authors conclude that the phosphodiesterase 4 inhibitor, rolipram, appears to have advantages over treatment
with antibodies to very late activating antigen-4 and interleukin-5 in mice rechallenged with allergen.
To determine whether or not an inhaled glucocorticoid can
prevent or reverse airway remodeling, Vanacker and coworkers (56) exposed rats to inhaled ovalbumin every second day
for two weeks. The animals developed an increase in the total
area of the airway wall, enhanced deposition of fibronectin,
proliferation of epithelial cells, hyperplasia of goblet cells, and
airway hyperresponsiveness. Administering inhaled fluticasone 30 minutes before each ovalbumin challenge produced a
decrease in all of the structural changes but it did not return
them to normal. Administering fluticasone for two weeks after
completing the ovalbumin challenge had no effect on the structural changes in the airways. The authors conclude that fluticasone partly prevents structural airway changes when given
simultaneously with ovalbumin but it does not reverse them.
To define the sequence of changes in inflammation and airway hyperresponsiveness after allergen challenge, Tomkinson
and coworkers (57) gave a single intranasal challenge of ovalbumin to previously sensitized BALB/c mice. Responsiveness
to methacholine showed a small increase at 8 hours, peaked at
24 to 48 hours, and had resolved by 96 hours. The neutrophil
infiltrate in bronchoalveolar fluid peaked at 8 hours and had
resolved by 48 hours. Eosinophils did not increase until 48 hours, they peaked at 96 hours, and remained elevated at 8 days; levels of eosinophil peroxidase were elevated only at 48 hours. Levels of tumor necrosis factor- peaked at 8 hours,
the levels of interleukin-4 and -5 peaked at 24 hours, and the
level of interleukin-13 was increased at both 24 and 48 hours.
Administration of an antibody to either interleukin-5, or very
late antigen-4 before the ovalbumin challenge, prevented the
development of airway hyperresponsiveness and the eosinophilia in bronchoalveolar fluid. The authors conclude that the
data identify the temporal association between cytokine production, eosinophil infiltration, and the development and resolution of airway hyperresponsiveness.
To determine the immediate response of small airways to
allergen challenge, Wohlsen and coworkers (58) put thin viable slices of lung into culture and measured airway constriction by video microscopy. The slices were taken from rats that
had been passively sensitized with serum from sensitized rats.
Exposure to ovalbumin produced an immediate allergic response. Both the extent (r = 0.74) and velocity (r = 0.49) of the
allergen-induced bronchoconstriction increased as the airway
size was decreased. The smaller airways also relaxed faster.
The bronchoconstriction was prevented by a serotonin receptor antagonist, ketanserin, but not by cyclooxygenase or lipoxygenase inhibitors, or by antagonists directed against histamine, acetylcholine, platelet-activating factor, or endothelin receptors. The authors conclude that the terminal bronchioles are more sensitive to allergen as compared with the larger airways, in part because of their increased sensitivity to serotonin.
To study the differing properties of mucous glycoproteins,
Shimizu and coworkers (59) induced hypertrophy and metaplasia in the goblet cells of the nasal epithelium by instilling
ovalbumin into the nose of ovalbumin-sensitized rats and by
instilling lipopolysaccharide into the nose of another group of
rats. Both challenges produced increases in mucin, but its
composition differed. After lipopolysaccharide, mucin contained 70% sulfomucin and 9% neutral glycoprotein. After ovalbumin, mucin contained 34% sulfomucin and 42% glycoprotein. Reactivity of the lectins, galactose-N-acetylgalactosamine and 2,3-linked sialic acid-galactose, was higher after lipopolysaccharide as compared with ovalbumin or saline.
Both lipopolysaccharide and ovalbumin caused a two-fold increase in the expression of messenger RNA for mucin. The
authors conclude that challenges with lipopolysaccharide and
ovalbumin produce similar increases in the expression of messenger RNA for mucin, but that the carbohydrate compositions of the newly produced mucin are different.
To define the importance of tumor necrosis factor- on the
development of allergen-induced airway hyperresponsiveness,
Kanehiro and coworkers (60) studied two groups of genetically manipulated mice and control mice. The mice were sensitized to ovalbumin and they subsequently received an airway
challenge with ovalbumin. Compared with control mice, the
mice genetically deficient in tumor necrosis factor- developed greater airway hyperresponsiveness, increased numbers
of eosinophils, and increased levels of interleukin-5 and interleukin-10 in bronchoalveolar fluid. SP-C/TNF--transgenic mice (which have 300 times higher levels of tumor necrosis
factor- in the lung as compared with transgene-negative
mice) failed to develop airway hyperresponsiveness, had lower
numbers of eosinophils, and lower levels of interleukin-5 and
interleukin-10 in bronchoalveolar fluid, as compared with transgene-negative mice. (SP-C/TNF-d-transgenic mice express tumor necrosis factor- in their lungs because of a transgene for
tumor necrosis factor- placed under the control of the promoter for surfactant protein C.) Depletion of T cells, which
are known to be activated by tumor necrosis factor- and to
negatively modulate airway hyperresponsiveness, produced
an increase in airway hyperresponsiveness in the transgenic
mice, but had no effect on the mice deficient in tumor necrosis
factor-. The authors conclude that tumor necrosis factor-
decreases airway hyperresponsiveness to allergen in mice and
that the action is mediated through activation of T cells.
Animal models: other challenges and mediators The M2 muscarinic receptors, which are located on the postganglionic parasympathetic nerves, inhibit the release of acetylcholine and vagally induced bronchoconstriction. Antigen challenge causes dysfunction of the M2 receptors in guinea pigs. To determine whether or not dexamethasone pretreatment would prevent M2 receptor dysfunction, Evans and coworkers (61) sensitized guinea pigs to inhaled ovalbumin. Pretreatment with dexamethasone before the antigen challenge prevented the hyperreactivity to vagal stimulation, the loss of M2 receptor function, and the recruitment of eosinophils to airway nerves, but it did not prevent the eosinophil influx into the airways. The authors conclude that dexamethasone prevents antigen-induced hyperreactivity by protecting M2 muscarinic receptors from antagonism by eosinophil major basic protein, and that this protection is achieved by specifically inhibiting the recruitment of eosinophils to airway nerves.
Elastase causes the release of tissue kallikrein (which cleaves kininogen to yield bradykinin) and bronchoconstriction in allergic sheep. Scuri and coworkers (62) determined whether or not hyaluronic acid, a large polysaccharide, inhibits this action of elastase. Inhaled porcine pancreatic elastase produced a 147% increase in pulmonary resistance and a 111% increase in the activity of tissue kallikrein in bronchoalveolar fluid. Pretreatment with inhaled hyaluronic acid blocked the release of tissue kallikrein and the bronchoconstriction in proportion to the dose and molecular weight. The authors conclude that hyaluronic acid blocks the bronchoconstriction induced by elastase in a dose-dependent and molecular weight dependent fashion, and that inhibiting the formation of kinins and the activity of tissue kallikrein contributes to this effect. To determine the role of apoptosis in the resolution of tissue eosinophilia by glucocorticoids, Uller and coworkers (63) induced lung edema and tissue eosinophilia by instilling Sephadex beads into the trachea of rats. Sephadex beads alone increased the total number of apoptotic cells, which were not efficiently engulfed by macrophages. Less than 0.3% of the eosinophils in lung tissue were apoptotic, whereas 20% of the eosinophils in the airway lumen were apoptotic and not engulfed. Intratracheal budesonide caused prompt resolution of lung edema, but it took three days of treatment with budesonide to reduce the tissue eosinophilia. Budesonide had no effect on apoptosis of tissue eosinophils. Eosinophils were eliminated by migration into the airway lumen where they underwent apoptosis. The authors conclude that apoptosis of eosinophils is exceedingly rare in lung tissue and is not influenced by glucocorticoids, and that the clearance of eosinophils is mediated by the migration of eosinophils into the airway lumen followed by apoptosis and mucociliary clearance.Ex-vivo studies
Because interleukin-13 and interleukin-4,
type 2 (Th2) helper cytokines, are believed to be important in
asthma, Laporte and coworkers (64) studied the effects of
these two cytokines on cultured smooth-muscle cells from the
human airway. Smooth-muscle cells expressed transcripts for
interleukin 4, interleukin 13 receptor 1 and interleukin 13 receptor II, but not for the chain of the interleukin 2 receptor. STAT-6, a transcription factor, was phosphorylated by
both interleukin-4 (peaking at 15 minutes) and interleukin-13
(peaking at 1 hour). Both interleukin-13 and interleukin-4 also
caused phosphorylation of extracellular signal-regulated kinase
mitogen-activated protein (ERK MAP) kinase. The -adrenergic responsiveness of the smooth-muscle cells was decreased
by interleukin-13, but not by interleukin 4. U0126, an inhibitor
of MEK (the enzyme that phosphorylates extracellular signal regulated kinase), reduced the effect of interleukin-13 on
-adrenergic responsiveness. The authors conclude that the
direct effect of interleukin-13 on smooth-muscle cells may
contribute to airway narrowing in patients with asthma.
. Interleukin-1 induced the release of several eosinophil-activating cytokines, including granulocyte-macrophage
colony-stimulating factor, RANTES and eotaxin. The release
of the cytokines was accompanied by phosphorylation of p42/
p44 extracellular signal-regulated kinases (ERKs), p38 mitogen-activated protein (MAP) kinase, and c-Jun amino-terminal kinase (SAPK/JNK). The release of eotaxin induced by interleukin-1 was inhibited by a specific inhibitor of p38 MAP kinase (SB 203,580) and by a specific inhibitor of p42/p44 ERK
(U 0126). The release of RANTES was inhibited only by U
0126. The release of granulocyte-macrophage colony-stimulating factor was inhibited by U 0126, and was enhanced by SB
203,580. The authors conclude that the release of eotaxin induced by interleukin-1 is regulated by pathways that involve both p38 MAP kinase and p42/p44 ERK, that release of
RANTES is dependent on activation of p42/p44 ERK and occurs independently of p38 MAP kinase activity, and that release of granulocyte-macrophage colony-stimulating factor is
dependent on p42/p44 ERK activation and is tonically suppressed by a mechanism that is partially dependent on p38
MAP kinase.
Because it is not known whether or not cytokines can modulate the expression of leukotrienes on airway smooth-muscle
cells, Amrani and coworkers (66) investigated the influence of
interferon- on the response of human airway smooth muscles
to leukotriene D4. Interferon- produced dose-dependent increases in messenger RNA and the expression of cysteinyl leukotriene receptor 1 on the surface of the smooth-muscle cells.
The effect of leukotriene D4 on cell stiffness, a proxy for force
development, was increased 5.4-fold by the addition of interferon-. Montelukast, an antagonist of cysteinyl leukotriene
receptor 1, completely inhibited the increase in cell stiffness
caused by leukotriene D4. Interferon- had no effect on the
cell stiffness response to bradykinin, another contractile agonist. The authors conclude that interferon- increases the responses of airway smooth-muscle cells to leukotriene D4 by
way of an increase in the expression of the cysteinyl leukotriene 1 receptor on the cell surface, and that this effect on
smooth muscle may contribute to airway hyperresponsiveness.
In human airway smooth-muscle cells, Accomazzo and
coworkers (67) studied the influence of variations in intracellular concentrations of calcium ion on bronchoconstriction
induced by leukotriene D4. Histamine and leukotriene D4
caused marked and equal constriction of strips of human
bronchi. In smooth-muscle cells, leukotriene D4, at variance
with histamine, elicited only a small transient change in intracellular calcium. The calcium-dependent, protein kinase C-
was activated by histamine, and to a lesser extent, by leukotriene D4, whereas only leukotriene D4 translocated the calcium-independent, protein kinase C- . Phorbol-dibutyrate ester, an activator of protein kinase C, caused contraction of
bronchial strips to the same extent in the presence and absence of calcium. In the absence of calcium, leukotriene D4
contracted the bronchial strips to the same extent as did the
activator of protein kinase C, suggesting involvement of calcium-independent, protein kinase C- . An inhibitor of protein
kinase C, H7, abolished the leukotriene D4-triggered contraction of bronchial strips in the absence of calcium, but the response was not greatly affected when calcium was present.
The authors conclude that leukotriene D4 contracts the human airway through a mechanism independent of the intracellular concentration of calcium ion and involves activation of
protein kinase C- .
Stimulation of the histamine H1-receptor leads to the formation of two secondary messengers: inositol triphosphate, which increases intracellular calcium, and diacylglycerol, which activates protein kinase C. Pype and coworkers (68) investigated
the effect of interleukin 1 on histamine-induced accumulation of inositol phosphate in human airway smooth-muscle
cells and the contractile response to histamine in human bronchial rings. Application of interleukin 1 for 24 hours caused
decreases in histamine-induced inositol phosphate formation
and bronchial contraction. An inhibitor of nuclear factor-
B
and an inhibitor of p38 mitogen-activated protein kinase
blocked the desensitization of the histamine H1-receptor caused by interleukin 1. Anisomycin, an activator of SAPK/
JNK and p38 mitogen-activated protein kinase, mimicked the
effect of interleukin 1. A cyclooxygenase antagonist, indomethacin, completely inhibited the effect of interleukin 1
on the histamine H1-receptor. Exogenous prostaglandin E2 desensitized the histamine H1-receptor. A selective inhibitor of
protein kinase A, 4-89, antagonized the effect of interleukin
1. The authors conclude that interleukin 1 protects airway
smooth muscle against histamine-induced inositol phospholipid hydrolysis and subsequent airway constriction by desensitizing the histamine H1-receptor.
Early and late asthmatic responses To determine whether or not inhaled glucocorticoids have a dose-dependent effect on airway responses to inhaled antigen, Inman and coworkers (69) did a double-blind crossover study of placebo versus three doses of mometasone furoate in 12 patients with mild asthma. The three doses (100, 200, and 800 µg daily) achieved equivalent reduction in the early asthmatic response and in allergen-induced hyperresponsiveness to methacholine. The late maximal fall in FEV1 was 24% after placebo, which was reduced by mometasone in a dose-dependent manner: 12% for 100 µg, 11% for 200 µg, and 6% for 800 µg. The increase in sputum eosinophilia was 60 (× 104 cells per ml) at 24 hours, and this was reduced by mometasone: 24 for 100 µg, 15 for 200 µg, and 6 for 800 µg. The authors conclude that inhaled mometasone furoate attenuates the early and late asthmatic responses, and that the detection of a dose-response effect for attenuating the late response may prove useful in evaluating the relative potency of inhaled glucocorticoids.
In 21 patients with mild asthma, Gauvreau and coworkers (70) compared the effects of inhaled cysteinyl leukotrienes E4 and D4 on airway inflammation. The maximum early fall in FEV1 was 29% after inhaling allergen, 24% after inhaling leukotriene D4, and 29% after inhaling leukotriene E4. Induced sputum revealed increases in eosinophils and basophils at 7 hours after a challenge with allergen and leukotriene E4, but not after challenge with leukotriene D4. Six patients revealed more eosinophils in the lamina propria after inhaling leukotriene E4 as compared with inhaling leukotriene D4. The authors conclude that for the same degree of bronchoconstriction, inhaled leukotriene E4 causes more tissue and airway eosinophilia as compared with leukotriene D4. To determine mechanisms by which inhaled mannitol causes bronchoconstriction, Brannan and coworkers (71) entered 20 patients with asthma into two separate double-blind studies. Pretreatment with fexofenadine hydrochloride, a histamine H1 receptor antagonist, decreased the sensitivity to mannitol to about one-third of the sensitivity obtained with placebo. The final reduction in FEV1 did not differ from pretreatment with placebo, and recovery of FEV1 was slower with fexofenadine. Pretreatment with the leukotriene antagonist, montelukast sodium, had no effect on the sensitivity to mannitol, and the final reduction in FEV1 did not differ from pretreatment with placebo. Recovery of FEV1 to baseline was faster with montelukast. The authors conclude that histamine contributes to the early airway response to inhaled mannitol and that leukotrienes contribute to sustaining and prolonging the response.Chemical and antigen challenge To determine whether or not a bronchial challenge with adenosine 5'-monophosphate (AMP) is more closely associated with airway inflammation as compared with methacholine, van den Berge and coworkers (72) studied 120 patients with asthma. In a stepwise multiple linear regression model, 18% of the variance in the PC20 for methacholine (the provocative concentration producing a 20% fall in FEV1) was explained by FEV1 (as percent predicted), and 23% of the variance was explained when peripheral blood monocytes, an independent predictor, were added to the model. In contrast, 25% of the variance in the PC20 for AMP was explained by the percentage of eosinophils in induced sputum, and 36% of the variance was explained when FEV1 (as percent predicted), an independent predictor, was added to the model. The authors conclude that a challenge with AMP more closely reflects airway inflammation as compared with a challenge with methacholine. An editorial commentary by Cockcroft (73) accompanies this article.
In patients with asthma, glucocorticoid therapy produces a greater improvement in the PC20 of AMP as compared with the PC20 of methacholine. To determine whether or not the different responses are related to airway inflammation, van den Berge and coworkers (74) tapered inhaled glucocorticoids in 120 patients with asthma and then made measurements before and after two weeks of treatment with glucocorticoids. Improvement in the PC20 of AMP was solely related to a reduction in airway inflammation. On multiple linear regression, the improvement in the PC20 of AMP was related (partial correlation coefficients) to sputum eosinophils (0.39), sputum lymphocytes (0.34), exhaled nitric oxide (0.29), and sputum
bronchial epithelial cells (0.19). The improvement was not
associated with an increase in percent predicted FEV1. Conversely, improvement in the PC20 of methacholine was correlated with a reduction in airway inflammation
sputum lymphocytes (0.31), sputum eosinophils (0.26)and with the
increase in percent predicted FEV1 (0.18). The total explained
variance of the improvement in bronchial hyperresponsiveness was greater for AMP as compared with methacholine (36 versus 22%). The authors conclude that the provocative concentration for AMP is more sensitive to changes in acute airway inflammation than is the provocative concentration for methacholine.
Hyperventilation- and exercise-induced asthma To determine whether or not nitric oxide is involved in the pathogenesis of thermally induced asthma, Kotaru and coworkers (75) studied 13 patients with asthma and 10 healthy subjects. The subjects performed isocapnic hyperpnea while breathing frigid air. Five minutes after hyperpnea, FEV1 fell by 28% in the patients with asthma and by 4% in the healthy subjects. During hyperpnea, the volume of exhaled nitric oxide rose in both groups, but the patients exhaled almost twice as much nitric oxide as compared with the control subjects. The increase in nitric oxide in the patients continued into the recovery period, and exhaled nitric oxide rose as airflow limitation developed. The authors conclude that nitric oxide plays an important role in thermally induced asthma.
Davis and Freed (76) delivered single challenges of cool dry air through a bronchoscope to specific bronchi of anesthetized dogs on five consecutive days. Peripheral airway resistance increased by 61% within 24 hours of the first challenge, by 113% on the third day, and stayed constant thereafter. The dry air challenges increased bronchial reactivity to hypocapnia and to intravenous histamine. The challenges produced increased levels of neutrophils, eosinophils, and leukotrienes in bronchoalveolar fluid. In the control dogs, intravenous albuterol produced a 46% decrease in airway resistance. In the challenged dogs, albuterol produced a decrease in resistance, although resistance was still 11% above baseline. The authors conclude that repeated dry air challenges cause peripheral airway inflammation, obstruction, hyperreactivity and impaired response to2-agonists.
Drugs
The effect of estrogen therapy in patients with
asthma is controversial. To investigate the mechanisms whereby
estrogen influences airway reactivity, Degano and coworkers
(77) compared the effect of physiologic doses of 17 -estradiol
versus placebo in oophorectomized female rats. In in vivo
studies, the airways of estrogen-treated rats had about one-sixth the responsiveness to inhaled acetylcholine as compared
with placebo-treated rats. In ex vivo studies of isolated tracheal segments, estrogen increased the contractile response to
acetylcholine but not the response to carbachol. The difference in contractile responsiveness between the estrogen and
placebo treated rats was abolished by either physostigmine, a
cholinesterase inhibitor, or by removing the epithelium. The
activity of acetylcholinesterase was 1.4 times greater in homogenates of the whole trachea from the estrogen-treated rats
than that of the placebo-treated rats. This difference disappeared when the epithelium of the trachea was removed. The
authors conclude that estradiol decreases the responsiveness
of the airways to acetylcholine and that the effect is partly
dependent on increased activity of acetylcholinesterase in the epithelium.
Other Pathophysiologic Mechanisms in Asthma
Tachykinins and neural activity The tachykinin, neurokinin A, is a powerful bronchoconstrictor that acts mainly on neurokinin 2 receptors. Amadesi and coworkers (78) determined whether or not activation of the neurokinin 1 receptor causes motor responses in isolated medium-size (2-5 mm) human bronchi. A selective agonist of the neurokinin 1 receptor, [Sar9, Met(O2)11]SP, contracted about 60% of the isolated bronchial rings. Two antagonists of the neurokinin 1 receptor, CP-999,994 and SR 140,333, reduced the contraction. The effect of the agonist was independent of the release of acetylcholine and histamine and their epithelial removal, and the contraction was not affected by inhibition of nitric oxide synthase and cyclooxygenase. The agonist caused increases in inositol phosphate, and these increases were blocked by SR 140,333 in medium and small-size (about 1 mm in diameter) bronchi and by a cyclooxygenase inhibitor in small but not in medium-size bronchi. The authors conclude that the neurokinin 1 receptors mediate bronchoconstriction in a large proportion of medium-size human bronchi, apparently through direct activation of smooth muscle receptors and release of inositol phosphate.
To study the role of sensory nerves in mediating lung inflammation caused by ozone, Graham and coworkers (79) studied two types of genetically modified mice. CCSP-NGF transgenic mice overexpress nerve growth factor from the lung-specific Clara cell secretory protein promoter and also exhibit hyperinnervation of the airway sympathetic and tachykinin-containing sensory nerve fibers. Exposure of these mice to ozone produced twice the number of neutrophils in bronchoalveolar fluid as compared with wild-type mice. The number of neutrophils in bronchoalveolar fluid was decreased by half in mice that are deficient in the gene for the low-affinity nerve growth factor receptor. In addition, administering neurokinin receptor antagonists reduced the level of neutrophilic inflammation in both the wild-type mice and the mice with overexpression of nerve growth factor. The authors conclude that the release of tachykinins from sensory nerves mediates the neutrophilic inflammation of the airways caused by ozone in mice. The autonomic control of the lower airway is primarily parasympathetic in nature. Because the cellular characteristics of neurons of the intrinsic ganglia of the human lower airway have not been reported, Kajekar and coworkers (80) made intracellular recordings from parasympathetic ganglia located on the bronchi of 39 human lungs. In response to a depolarizing current step, tonic neurons responded with repetitive and sustained action potentials. Phasic neurons generated one action potential and then accommodated. Phasic neurons were differentiated as having after-hyperpolarizing potentials that followed a single action potential, which were of either short or long duration. In phasic neurons, stimulation of preganglionic nerves elicited one or two populations of nicotinic fast excitatory postsynaptic potentials, which were graded in amplitude, subthreshold for action potential generation, and decreased in amplitude during higher frequency stimulation. In tonic neurons, single preganglionic stimuli elicited two to five populations of fast excitatory postsynaptic potentials, and one to three of these were at threshold for action potential generation. The authors conclude that neurons located in human bronchial ganglia have anatomic, synaptic, and membrane properties that are indicative of an integrative function. Widdicombe (81) recalls an early study on the neural pathways involved in cough.Deep inspiration A deep inhalation both reverses and prevents against bronchoconstriction in healthy subjects, but the bronchoprotector effect is impaired in patients with asthma. To determine whether or not the impaired bronchoprotector effect is related to the degree of bronchial hyperresponsiveness, Scichilone and coworkers (82) studied 10 healthy subjects, 12 patients with asthma and moderate-to-severe bronchial hyperresponsiveness, 14 patients with asthma and mild-to- borderline hyperresponsiveness, and 10 patients with allergic rhinitis and mild-to-borderline hyperresponsiveness. In the absence of a deep inhalation, a single challenge with methacholine produced equivalent decreases in FEV1 in the four groups. Taking a deep inhalation before methacholine had a bronchoprotector effect only in the healthy subjects. Taking a deep inhalation after methacholine had a bronchodilator effect in all four groups, and the effect was strongest in the healthy subjects. The authors conclude that the failure of a deep inhalation to protect against bronchoconstriction is related to bronchial hyperresponsiveness rather than to a clinical diagnosis of asthma, and that the dissociation between bronchoprotection and bronchoconstriction suggests that the two effects involve different mechanisms.
In eight patients with asthma, King and coworkers (83) determined whether or not prohibiting deep inhalation in the course of a methacholine challenge would affect the bronchoconstrictor response. When the patients inhaled four doses of a PC15 concentration of methacholine (the concentration producing a 15% fall in FEV1) every 5 minutes, the decrease in FEV1 was greater when deep inhalations were prohibited as compared with when deep inhalations were allowed (36 versus 21%). The same pattern was seen after inhaling five doses of methacholine (39 versus 19%). The authors conclude that prohibiting deep inhalation increases the bronchoconstrictive response to methacholine in patients with asthma. In 10 patients with asthma and nine healthy subjects, Brown and coworkers (84) used high-resolution computed tomography to examine the ability of a deep inspiration to distend the airways. Both at baseline and after increasing airway tone with methacholine, a deep inspiration produced similar distention of the airways of the patients and of the healthy subjects. After inducing an increase in airway tone with methacholine and after performing a deep inspiration, bronchodilation occurred in the healthy subjects whereas further bronchoconstriction occurred in the patients. The authors conclude that a deep inspiration causes similar distension of the airways in patients with asthma and healthy subjects, but after induced bronchoconstriction the patients with asthma develop bronchoconstriction after a deep inhalation and the healthy subjects develop bronchodilation. Because studies of the bronchodilator and bronchoprotective effect of a deep inhalation have relied on spirometry, Lutchen and coworkers (85) measured resistance and elastance between 0.1 and 8 Hz in 12 patients with asthma and 7 healthy subjects. In general, prohibition of a deep inhalation increased hyperresponsiveness to methacholine. In patients with mild-to-moderate asthma and in healthy subjects, the pattern o