Endobronchial Allergen Challenge Research Articles

Cellular changes 24 hours after endobronchial allergen challenge in asthma.

Cellular changes 24 hours after endobronchial allergen challenge in asthma.

The airway inflammatory response in allergic asthma and its relationship to clinical disease

Endobronchial biopsy and lavage studies have revealed the presence of mast cell, eosinophil, T-lymphocyte and epithelial cell activation in asthma, along with the structural changes of tissue eosinophil infiltration, loss of superficial columnar ciliated epithelial cells and enhanced collagen deposition in the laminar reticularis. As these cellular and structural changes underlie the clinical features of asthma, i.e., symptom expression, variable airflow obstruction and bronchial hyperresponsiveness, and understanding of their induction and regulation is essential to the understanding of the asthmatic process. The acute airway response to allergen has been studied by the technique of local endobronchial allergen challenge with direct airway sampling in asthma. These studies identify allergen-mast cell interaction as the initial airway event, with mediator release inducing bronchoconstriction and enhancing vascular permeability. As preformed cytokines are present in mast cells, cytokine release from this cell population is likely to initiate the process of endothelial cell activation, with upregulation of cell adhesion molecules, and tissue cell recruitment. Subsequent cytokine elaboration from airway macrophages and T-lymphocytes will perpetuate this response while in chronic clinical disease T-lymphocytes, mast cells, matrix tissue, epithelial cells and eosinophils themselves are all likely to contribute to the cytokine pool within the airways and thus to the regulation of inflammatory cell migration and activation.

Bronchial biopsy evidence for leukocyte infiltration and upregulation of leukocyte-endothelial cell adhesion molecules 6 hours after local allergen challenge of sensitized asthmatic airways.

We have examined the mucosal changes occurring in bronchial biopsies from six atopic asthmatics 5-6 h after local endobronchial allergen challenge and compared them with biopsies from saline-challenged segments from the same subjects at the same time point. All the subjects developed localized bronchoconstriction in the allergen-challenged segment and had a decrease in forced expiratory volume in 1 s (FEV1) (P < 0.01) and a decrease in their methacholine provocative concentration of agonist required to reduce FEV1 from baseline by 20% (P < 0.05) 24 h postchallenge. At 6 h we observed an increase in neutrophils (P = 0.03), eosinophils (P = 0.025), mast cells (P = 0.03), and CD3+ lymphocytes (P = 0.025), but not in CD4+ or CD8+ lymphocyte counts. We also detected an increase in endothelial intercellular adhesion molecule type 1 (P < 0.05) and E-selectin (P < 0.005), but not vascular cell adhesion molecule type 1 expression with a correlative increase in submucosal and epithelial LFA+ leucocytes (P < 0.01). Thus, in sensitized asthmatics, local endobronchial allergen instillation leads to an increased inflammatory cell infiltrate of the airway mucosa that involves upregulation of specific adhesion molecules expressed on the microvasculature.

Eosinophils express interleukin 5 and granulocyte macrophage-colony-stimulating factor mRNA at sites of allergic inflammation in asthmatics.

IL-5 and granulocyte macrophage-colony-stimulating factor (GM-CSF) are important regulators of eosinophil survival, proliferation, and effector function. To determine whether IL-5 and/or GM-CSF are generated by eosinophils at sites of allergic inflammation, we have used in situ hybridization with 35S-labeled RNA probes to study the expression of IL-5 and GM-CSF mRNA in bronchoalveolar lavage (BAL) eosinophils derived from asthmatics (n = 5) before and after endobronchial allergen challenge. Endobronchial allergen challenge induced a significant airway eosinophilia (pre-allergen challenge 0.6 +/- 0.5% eosinophilia vs post-allergen challenge 48.2 +/- 25.6% eosinophilia). Post-allergen challenge eosinophils expressed IL-5 and GM-CSF mRNA, but did not express IL-1 beta or IL-2 mRNA. To determine whether the IL-5 mRNA-positive cells coexpressed GM-CSF mRNA, double mRNA labeling experiments with a digoxigenin-11-UTP nonradioactive labeled IL-5 RNA probe and a GM-CSF 35S-labeled RNA probe were performed. These studies demonstrated that individual eosinophils expressed one of four cytokine mRNA profiles (IL-5+, GM-CSF+, 34 +/- 13%; IL-5+, GM-CSF-, 34 +/- 5%; IL-5-, GM-CSF+, 11 +/- 9%; IL-5-, GM-CSF-, 21 +/- 25%). The expression of IL-5 and GM-CSF by eosinophils at sites of allergic inflammation in asthmatics may provide an important autocrine pathway, maintaining the viability and effector function of the recruited eosinophils.

Elevation of tissue kallikrein and kinin in the airways of asthmatic subjects after endobronchial allergen challenge.

Bronchial tissue kallikrein is the major kininogenase activity in the airways of asthmatic subjects. The relationship of IgE-mediated events to its release and/or activation is unknown, however, and is the subject of this report. Seven subjects with mild atopic asthma underwent endobronchial challenge with relevant aeroallergen. Baseline pre-allergen lavage and sequential post-challenge lavages were collected over an approximate 10-minute time course. Individual aliquots were analyzed separately and compared with saline control lavages performed in a separate lobe. In five of the seven subjects, an increase in tissue kallikrein activity, measured by cleavage of the synthetic substrate Val-Leu-Arg-pNA, was identified in the post-challenge lavages. The antigenic identity of the enzymatic activity was confirmed as a tissue kallikrein in each case by immunoblotting. Tissue kallikrein activity was highly correlated with the appearance of immunoreactive histamine and kinin (p = 0.0001). High molecular weight kininogen influx and cleavage was detected in the post-challenge samples by immunoblotting and paralleled the detection of kinin in BAL fluid. Two of the subjects, despite clinical profiles similar to those of the five positive responders, failed to react to endobronchial challenge. Saline control lavages contained detectable kallikrein, kinin, and histamine in two subjects; in each case, however, this was significantly less than in the post-allergen samples. The results demonstrate a close association between immediate type hypersensitivity events in the lower airway and the appearance of active kallikrein, kininogen substrate, and the liberation of kinin.

Endobronchial allergen challenge in asthma. Demonstration of cellular source of granulocyte macrophage colony-stimulating factor by in situ hybridization.

Airway inflammation is thought to play an important role in the pathogenesis of asthma. We have used in situ hybridization and an immunoassay to determine whether granulocyte macrophage colony-stimulating factor (GM-CSF) (a cytokine capable of eosinophil activation) is present in the airway of asthmatics (n = 6) who have 37.0 +/- 15.1% airway eosinophilia after endobronchial allergen challenge. Levels of immunoreactive GM-CSF (less than 4 pg/ml pre-allergen versus 180.5 +/- 46.9 pg/ml post-allergen) increased significantly 24 h after endobronchial allergen stimulation. The cellular source of bronchoalveolar lavage (BAL) GM-CSF, as determined by in situ hybridization and immunoperoxidase staining, was derived predominantly from UCHL-1 positive BAL lymphocytes, as well as from a smaller population of alveolar macrophages. Before local endobronchial allergen challenge, less than 1% of lymphocytes and alveolar macrophages recovered by BAL expressed GM-CSF mRNA, whereas after allergen stimulation 92.6 +/- 3.4% of lymphocytes and 17.5 +/- 22.7% of alveolar macrophages expressed GM-CSF mRNA. This study provides evidence that in an experimental model of allergen-induced asthma, activation of the immune and inflammatory response (BAL lymphocyte and alveolar macrophage production of GM-CSF) is temporally associated with an inflammatory cell influx of eosinophils into the airway.

Tryptase, a mediator of human mast cells

Tryptase, a mediator secreted by human mast cells during immediate reactions, has demonstrated effects on several pathways in vitro. This enzyme can rapidly inactivate fibrinogen and, as a complex with heparin, may prevent coagulation that may otherwise occur when plasma enters tissues at sites of immediate reactions. Tryptase may also activate prostromelysin, which in turn activates latent collagenase. When canine pulmonary smooth muscle is incubated with canine tryptase, the contractile response to histamine is increased. Tryptase, quantifiable in complex biologic fluids by immunoassay, can serve as a specific indicator of mast cell involvement in certain clinical settings. For example, after bee sting--induced anaphylaxis, tryptase levels in the blood peak at approximately 1 hour, then decline with a half-life of approximately 2 hours. Additionally, elevated tryptase levels in bronchoalveolar lavage fluid of asymptomatic, atopic persons with asthma suggest ongoing mast cell activation, which may relate to adenosine hyperresponsiveness and a persistence of bronchial hyperreactivity. Tryptase levels in bronchial lavage fluid of atopic patients with asthma rise markedly after endobronchial allergen challenge but not after an exercise challenge, suggesting a lack of mast cell involvement in the latter condition.

Elevated Levels of Leukotriene C4in Bronchoalveolar Lavage Fluid from Atopic Asthmatics after Endobronchial Allergen Challenge

Sulfidopeptide leukotrienes have been implicated in the pathogenesis of asthma because of their ability to induce bronchospasm, airways hyperreactivity, and increased mucus production. In the present study, the leukotrienes (LT) C4, D4, and E4 were measured in bronchoalveolar lavage fluid (BALF) before and 5 min after endobronchial allergen challenge in four subject groups: nonatopic nonasthmatic, nonatopic asthmatic, atopic nonasthmatic, and atopic asthmatic. As determined by high performance liquid chromatography (HPLC), after allergen challenge, the predominant sulfidopeptide leukotriene found in BALF from atopic asthmatics was LTC4. Smaller amounts of LTD4 and LTE4 were detectable. The baseline level of leukotrienes in the atopic asthmatics was 64 +/- 18 pg/ml, with measurable levels being found in nine of 11 samples. Atopic nonasthmatics had measurable levels in only one of seven baseline samples, whereas five of six nonatopic subjects had undetectable levels. Allergen challenge in atopic asthmatics resulted in significant increases in LTC4 over prechallenge levels (64 +/- 18 to 616 +/- 193 pg/ml) (p less than 0.01) and over levels in the three control groups after challenge (p = 0.0297). The atopic nonasthmatic group also had detectable leukotriene levels after allergen challenge (88 +/- 32 pg/ml), whereas leukotrienes remained undetectable in five of the six nonatopic samples. For comparison, histamine and the prostanoids prostaglandin D2 (PGD2) and thromboxane B2 (TxB2) were also measured in BALF. The levels of all three of these mediators increased in BALF from atopic asthmatics after allergen challenge. After allergen challenge, the best correlation was found between the levels in BALF for the prostanoids PGD2 and TxB2 (r = 0.88).(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of Pulmonary Mast Cells by Bronchoalveolar Allergen Challenge:In VivoRelease of Histamine and Tryptase in Atopic Subjects with and without Asthma

Human mast cells likely play a significant role in human asthma. In the present study, concentrations of tryptase and histamine in bronchoalveolar lavage fluid (BALF) were used as indicators of pulmonary mast cell activation. BALF was obtained before and after endobronchial allergen challenge and assessed for mediator content and cell composition in 4 subject groups: nonatopic nonasthmatics (Group 1, n = 7), nonatopic asthmatics (Group 2, n = 3), atopic nonasthmatics (Group 3, n = 7), and atopic asthmatics (Group 4, n = 7). Before challenge, histamine concentrations were not different between the 4 groups, whereas tryptase concentrations were significantly greater in the atopic asthmatics than in each of the other groups (p less than 0.04). Allergen challenge in atopic asthmatics resulted in significant increases above baseline in mean +/- SD histamine (0.7 +/- 7.1 to 2.8 +/- 2.0 ng/ml) and tryptase (2.0 +/- 1.7 to 10.1 +/- 8.2 ng/ml) concentrations in BALF (p less than 0.03). Atopic nonasthmatics also had increases above baseline in histamine (0.2 +/- 0.2 to 1.2 +/- 1.4 ng/ml) and tryptase (0.5 +/- 0.4 to 1.4 +/- 1.03 ng/ml) concentrations after allergen challenge (p less than 0.05). Though the histamine values were not significantly different between atopic nonasthmatics and atopic asthmatics after allergen challenge, tryptase concentrations were markedly higher in the atopic asthmatic group. The numbers, as well as the predominance of the T mast cell type in atopic asthmatics and nonasthmatics, were no different from controls. In nonatopic subjects, regardless of asthmatic state, histamine or tryptase concentrations were not altered by allergen challenge.(ABSTRACT TRUNCATED AT 250 WORDS)