D Vanhee

Modulation of cell adhesion molecules on human endothelial cells by eosinophil-derived mediators.

Mechanisms that allow a selective eosinophil emigration in different eosinophilic lung diseases remain poorly understood. In this study, we tested the hypothesis that eosinophils might participate in their own recruitment, particularly through adhesion molecule expression on human endothelial cells (EC). Blood eosinophils from donors with blood eosinophilia were purified and maintained in culture medium for 1 and 18 h. The expression of ICAM‐1, E‐selectin, and VCAM‐1 on human umbilical vein endothelial cells (HUVEC) was evaluated by ELISA and flow cytometry analysis after addition of eosinophil supernatants. Eosinophil supernatants collected after 1 h induced a weak increase of CAM expression on HUVEC. In contrast, supernatants from eosinophils cultured for 18 h considerably amplified ICAM‐1, E‐selectin, and VCAM‐1 expression on the surface of EC. These levels of CAM expression (in optical density determined by ELISA) were about two‐ or threefold more important than those obtained with eosinophil supernatants collected after a 1‐h culture. The characterization of the implicated molecules showed that anti‐IL‐1β antibodies significantly inhibited ICAM‐1, E‐selectin, and VCAM‐1 expression, whereas anti‐TNF‐α antibodies only induced a moderate inhibition. Our data support the hypothesis that eosinophils, through the release of at least IL‐1β and TNF‐α, might participate in the amplification of the inflammatory reaction by activating the vascular endothelium. J. Leukoc. Biol. 63: 351–358; 1998.

A colorimetric assay to evaluate the immune reactivity of blood platelets based on the reduction of a tetrazolium salt.

Since the first report in 1983, blood platelet reactivity from patients with immediate hypersensitivity reactions, and particularly allergic asthma and aspirin-sensitive asthma, has been evaluated by the release of cytotoxic mediators able to induce the death of parasite larvae, and by the generation of oxygen derived free radicals, measurable by chemiluminescence. We demonstrate here that platelets are able to reduce MTT tetrazolium salt proportionally to the stimulation level in two models of platelet triggering, one IgE-dependent and one aspirin-dependent. This method correlated significantly with the cytotoxicity assay of platelet stimulation (r = 0.965, p < 10(-5) for IgE-dependent stimulation; r = 0.723, p < 10(-4) for aspirin-dependent stimulation). The MTT colorimetric assay should complement or possibly replace previous methods of assessing platelet activation which were of limited use allowing broader investigations on the involvement of platelets in immune processes and in allergic or pseudoallergic reactions.

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.