Ph. Gosset

STIMULATION OF ALVEOLAR MACROPHAGES IN ASTHMATIC PATIENTS AFTER LOCAL PROVOCATION TEST

Dermatophagoîdes pteronyssinus allergen was instilled into the alveolar space of 7 allergic asthmatic patients during bronchoalveolar lavage. beta-glucuronidase concentration in the bronchoalveolar fluid from the challenged lung became significantly higher than that in the opposite control lung (3.90 +/- 1.88 nmol/h versus 0.86 +/- 0.55). The intracellular level of beta-glucuronidase in the alveolar macrophages in the challenged lung was 40.3% lower than that in the control lung. In 3 controls intracellular concentrations of beta-glucuronidase were similar before and after allergenic challenge and no enzyme activity could be detected in bronchoalveolar lavage fluid. These results suggest that alveolar instillation of an allergen in asthmatic patients rapidly stimulates alveolar macrophages, which may be involved in immediate hypersensitivity reactions in asthma.

Interactions Between Endothelial Cells and Effector Cells in Allergic Inflammation

The local inflammatory response that occurs after repeated exposure to allergens or during the late-phase reaction results from a complex network of interactions between inflammatory cells (mast cells, eosinophils, macrophages) and resident cells belonging to the lung structure itself like EC, fibroblasts, or bronchial epithelial cells. Among structural cells, EC represent critical elements: they control leukocyte traffic through the expression of adhesion molecules; they are also able to amplify leukocyte activation through the production of proinflammatory cytokines like IL-1, IL-6, or of chemokines like IL-8. Three cell models have been successively considered. When supernatants of alveolar macrophages, recovered from patients exhibiting a late asthmatic response after allergen exposure, were tested on HUVEC cultures, a TNF alpha-dependent ICAM-1 and E-selectin overexpression was observed. Among mast-cell mediators, histamine was already known to induce a rapid and transient expression of P-selectin; we demonstrated that histamine also induced an IL-6 and IL-8 secretion by HUVEC, which was concentration-dependent and inhibited by H1 or H2 receptor antagonists. Finally purified eosinophils obtained from donors with hypereosinophilia similarly increased adhesion molecule expression and chemokine production. The precise nature of the eosinophil product(s) involved in this process is currently under investigation.

Potential Implication of Endothelial Cells in Bronchial Asthma

Inflammatory cells like eosinophils, neutrophils or mononuclear phagocytes have long been recognized as essential components in the pathophysiology of asthma. After recruitment in situ and subsequent activation, they are considered as responsible for epithelial and submucosal bronchial alterations. However, to access to the inflammatory site, these cells have to cross the endothelial wall, suggesting so a potential implication of endothelial cells (EC) in bronchial asthma. To test this hypothesis, we studied in a first step the modulation of vascular adhesions like intercellular adhesion molecule-1 (ICAM-1) on EC: supernatants of alveolar macrophages (AM) recovered by bronchoalveolar lavage in patients exhibiting a late asthmatic reaction, induced an enhanced expression of ICAM-1 on EC preparations; increase of ICAM-1 was clearly correlated to amounts of tumor necrosis factor-alpha (TNF alpha) present in AM supernatants, as shown by inhibition experiments with anti-TNF alpha antiserum. The second way to explore the possible role of EC in asthma was the detection of autoantibodies to EC in various allergic disorders: antibodies against a 120-kD EC antigen in patients with allergic granulomatosis and angiitis, antibodies towards a 55-kD component, common to human EC and platelets in patients with severe asthma, namely characterized by their corticosteroid dependence or by aspirin-induced intolerance. So our data suggest that bronchial asthma might result from either EC activation, through the induction of surface adhesion molecules or from an autoimmune process involving EC antigens.

In vitro assessment of environmental toxicology using alveolar cells astarget

Biphasic culture of alveolar cells (alveolar macrophages and type II cells) has been widely developed and permits a precise evaluation of the toxic effects of air pollutants. Clearly, in vitro exposure of alveolar cells to high concentrations of oxidant gases is responsible for a loss of cell viability. In contrast, when exposed to realistic concentrations of gases (NO2, O3), cell viability is not altered and various proinflammatory mediators are released. This in vitro model has proved to be sensitive at levels of gas exposure of ambient air quality standards and appears a sensitive biological indicator of air pollutant cell toxicity.

Experimental systems for mechanistic studies of toxicant induced lung inflammation

Human breath contains a large array of complex and poorly characterized mixtures. We can measure the potential risk of these exposures at molecular, cell, organ, organismic levels or in population. This paper emphasizes the characteristics of in vitro tests of lung cells and discusses the use of in vitro systems to determine the health effects of inhaled pollutants. Exposure to gases can be performed with roller bottles fitted with modified rotating caps with tubing connections, or by using dishes on rocker platforms, which tilt back and forth to expose the cell culture to gases. Exposure of cells may also be obtained by using very thin gas-permable membrane on which cells grow. However, it is clear that in using these systems, the culture medium constitutes a barrier between the gas and the target cells and thus does not permit a physiological approach of the toxic effects of gases. This is the reason why an experimental model, using a biphasic cell culture technique in gas phase, was developed. We report the value and the limits of this method using bronchial cells or alveolar macrophages. Exposure of lung cells to gas pollutants or particles may be responsible for either cell injury or cell activation associated with the overexpression of mRNA and the release of various bioactive mediators. In vitro assays have some limitations, particularly because the human pulmonary response to inhaled pollutants is the result of complex interactions involving many different cell types within the lungs. However, cell culture using biphasic systems in aerobiosis opens new ways for the research on the biological effects of gas pollutants.

Alveolar Macrophages Secretory Dysfunctions in Coal Workers’s Pneumoconiosis. Comparison Between Simple Pneumoconiosis and Progressive Massive Fibrosis.

Coal worker’s pneumoconiosis (CWP) comprises a variety of pulmonary radiological and pathological changes resulting from the inhalation of coal dust. Coal worker’s pneumoconiosis is usually divided in two groups: simple pneumoconiosis (SP) and progressive massive fibrosis (PMF). This distinction is of importance in clinical practice since PMF is definitively disabling. However, why some workers should develop PMF when exposed to dust concentrations similar to those experimencied by workers who develop only SP is unknown. Current concepts emphazise the role of alveolar macrophages in the pathogenesis of chronic interstitial lung diseases (Voisin, 1983; Davis, 1986). In this way, the cells recovered by bronchoalveolar lavage were extensively used to evaluate the distribution of cell type and the secretory activities of alveolar macrophages that could be relevant to the pathogenesis of CWP, such as superoxide anion, neutrophil chemotactic factors, fibronectin (Wallaert, 1986). Here we have extended our study on macrophage secretory dysfunctions by the measurement of interleukin-1 and tumor necrosis factor alpha spontaneously released by alveolar macrophages.

Evaluation of Proinflammatory Cytokine Expression in Alveolar Macrophages and in the Lung of Pneumoconiotic Patients

In order to confirm their in vivo participation in the pathogenesis of Coal Workers’ Pneumoconiosis (CWP), TNF and IL-6 secretion and expression were evaluated in bronchoalveolar lavage (BAL) and lung tissue from pneumoconiotic patients. BAL was collected from 19 simple pneumoconiosis (SP) and 8 progressive massive fibrosis (PMF), while lung parenchyma specimen were obtained from 1 SP and 3 PMF. TNF and IL-6 mRNA expression was assessed using in situ hybridization. To identify the cellular source of these monokines, immunocytochemistry was performed on lung tissue. TNF and IL-6 concentrations were quantified using bioassay. In situ hybridization was performed using 35S-labelled RNA probes encoding for TNF and IL-6.