M. Bachelet

Increased spontaneous release of tumour necrosis factor-alpha by alveolar macrophages from wheezy infants

We determined if alveolar macrophages (AMs) from infants with severe recurrent wheezing episodes release increased amounts of tumour necrosis factor-alpha (TNF-alpha), as described in adults with asthma. We compared TNF-alpha release by unstimulated and lipopolysaccharide-stimulated AMs obtained by bronchoalveolar lavage in 13 wheezy and seven nonwheezy infants (aged 6-36 months) and analysed its regulation by dexamethasone. Metabolites in cell supernatants were quantified by enzyme-linked immunosorbent assay (ELISA) (TNF-alpha) or radioimmunoassay (thromboxane B2 and prostaglandin E2). Comparison of results was performed by the Mann-Whitney U-test and values were expressed as median (interquartile range) in ng x 10(6) cells(-1). Resting AMs from wheezy infants released larger amounts of TNF-alpha and thromboxane B2 as compared to controls: 2.67 (0.89-8.33) vs 0.48 (0.25-1.08) and 75.63 (38.07-158.91) vs 10.03 (7.36-76.08), respectively (p<0.05). When stimulated overnight with bacterial lipopolysaccharide, AMs from both groups released similar amounts of metabolites. Dexamethasone induced a consistent inhibition of the lipopolysaccharide-stimulated release of all the mediators. Our results show that alveolar macrophages from wheezy infants are activated to release increased amounts of tumour necrosis factor-alpha, as in asthma, and suggest that infants with recurrent wheezing may eventually benefit from treatment with glucocorticoids.

Increased eosinophil cationic protein levels in bronchoalveolar lavage from wheezy infants

Although studies examining the serum suggest a role for eosinophils in wheezing episodes in infants and toddlers, the presence of a chronic eosinophilic inflammation within their airways remains to be demonstrated. In this study we investigated whether eosinophil cationic protein (ECP) levels are increased in BAL fluid (BALF) from infants and toddlers with recurrent wheezing episodes, during an asymptomatic period. The levels of ECP in BALF were quantitated by radioimmunoassay in 61 children (36 with severe recurrent episodes of wheezing and 25 who were non‐wheezy), aged 6–36 months, in whom flexible bronchoscopy was clinically indicated. BALF eosinophil counts were ≤ 1% in all patients and did not differ in wheezers, compared to non‐wheezers. In contrast, ECP levels in BALF were ≥ 2.2 µg/l in 18 of 36 (50%) wheezy infants but in only three of 25 (12%) control infants (p < 0.01). Neutrophil counts were significantly higher in the wheezer group than in the non‐wheezer group (8.1 × 103 cells/ml vs. 3.0 × 103 cells/ml). ECP levels in the BALF were not correlated with the absolute number of eosinophils (r = 0.03; p = 0.8) but were correlated with the absolute number of neutrophils (r = 0.54; p = 0.001). There was no association between high ECP levels in BALF and the atopic status of the wheezers. In conclusion, ECP levels are increased in BALF from young children with recurrent wheezing episodes, even during relatively quiescent periods, suggesting a chronic increased cell activation in the lower airways.

Immunoglobulin-G-dependent stimulation of guinea pig lung mast cells and macrophages

Alveolar macrophages and mast cells isolated from guinea pig lung were passively sensitized with IgG1, IgG2, or serum obtained from guinea pigs actively sensitized with ovalbumin. The release of histamine by mast cells and of thromboxane A2 by alveolar macrophages upon ovalbumin challenge indicated that both antibodies and serum were capable of sensitizing these cells with similar effectiveness. Heating the scrum at 56°C for 4 h to inactivate IgE did not modify the antigen‐dependent response of lung cells. These results suggest a predominant role for IgG in the allergic response of the guinea pig through the activation of different cell types such as lung mast cells and alveolar macrophages.

Reduced responsiveness of bronchoalveolar cells from sensitised guinea-pigs to the cyclic AMP—Stimulating effect of prostaglandin E2 and β-adrenoceptor agonists

Prostaglandin E2 and the beta-adrenoceptor agonist salbutamol were less effective in increasing the intracellular cAMP content of bronchoalveolar cells and adherent alveolar macrophages from ovalbumin-sensitised as compared to control guinea-pigs. This refractoriness to the cyclic AMP-stimulating effects of PGE2 and salbutamol induced by the sensitisation procedure may be relevant to the altered bronchopulmonary responsiveness in asthma.

PAF-acether and experimental anaphylaxis as a model for asthma.

Several lines of evidence indicate that platelet-activating factor (PAF-acether) is implicated in hypersensitivity reactions. Indeed, PAF-acether reproduces the features of asthma in vivo and in vitro, since it induces bronchoconstriction, hypotension, and hemoconcentration and activates platelets and leukocytes. Both PAF-acether and antigen evoke eosinophil margination and diapedesis in guinea pig lung parenchyma and bronchial submucosa. Furthermore, intradermal administration of PAF-acether to atopics induces a more intense eosinophil degranulation as compared to normal subjects. PAF-acether also induces bronchopulmonary hyperresponsiveness in various animal models and in humans. We showed that lungs from actively sensitized guinea pigs exhibit an in vitro bronchopulmonary hyperresponsiveness to PAF-acether as compared to nonsensitized animals. This phenomenon is probably due to a lung invasion by inflammatory cells or to a variation of the reactivity of resident lung cells such as alveolar macrophages. In these cells, the cyclic adenosine monophosphate content is much less increased by prostaglandin E2 and salbutamol when they are obtained from actively sensitized animals.

5-Lipoxygenase and endotoxin-induced microvascular albumin exchanges and leucocyte recruitment in guinea-pig lungs

The interference of the 5-lipoxygenase inhibitor, BW B70C ((E)-N-(3-[3-(4-fluorophenoxy)phenyl]-1(R,S)-methyl prop-2-enyl)-N-hydroxyurea), with Escherichia coli lipopolysaccharide (endotoxin)-induced lung leucocyte sequestration and microvascular albumin exchanges was evaluated in the anaesthetised guinea-pig using radioactive tracers, in parallel to the effects on cell counts in the broncho-alveolar lavage fluid, blood tumour necrosis factor (TNF-alpha) content, secretion of phospholipase A2 and synthesis of leukotriene C4 by alveolar macrophages. Intravenous injections of 0.1 or 1 mg/kg endotoxin induced lung leucocyte sequestration but only the higher dose induced an increase in albumin microvascular exchanges and the infiltration of leucocytes towards the airway lumen. Leukotriene B4, a potential mediator of the 5-lipoxygenase-dependent endotoxin effects, induced a rapid and transient lung leucocyte sequestration and leucopenia associated with a more progressive increase in microvascular exchanges. The 5-lipoxygenase inhibitor, BW B70C, injected i.p. (30 mg/kg) prevented leukotriene C4 synthesis by alveolar macrophages and reduced leucocyte migration to the airways lumen as well as albumin microvascular leakage but did not affect the endotoxin-induced increase in the blood level of TNF-alpha and of secreted phospholipase A2. However, BW B70C failed to modify vascular leucocyte margination induced by 1 mg/kg endotoxin, suggesting that, apart from a role of 5-lipoxygenase, alternative pathways operate in response to endotoxin in guinea-pig.

Interactions between cyclic AMP stimulating drugs and PAF-acether in guinea-pig alveolar macrophages

Intra-tracheal administration of PAF-acether (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) to guinea-pigs induces a bronchoconstriction, which appears to depend on the cell population located in the bronchi, mainly alveolar macrophages [1]. In addition, the exposure of alveolar macrophages in vitro to PAF-acether results in an increased liberation of arachidonic acid (AA) from phosphatidylcholine and phosphatidyl-inositol, suggesting the activation o fPLA 2 or PLC in concert with diglyceride lipase activities [2]. Activation of macrophages is known to be suppressed by PGE 2 and /r agonists because of their ability to elevate intracellular cAMP (cyclic AMP) concentrations [3]. Considering these findings, and in view of the hypothetized role of PAF-acether and alveolar macrophages in asthma, we examined the possible involvement of cAMP levels for controlling the effects of PAF-acether in alveolar macrophages.

Passive sensitization of guinea-pig lung mast cells and alveolar macrophages with anti-ovalbumin IgG1 and IgG2 antibodies

In the present study, we performed experiments to explore the role of IgG immunoglobulins in type I hypersensitivity reactions in the guinea-pig. Our results suggest that IgG immunoglobulins may be involved in allergic reactions, both through histamine release from mast cells and thromboxane A2 release from macrophages after challenge of passively sensitized cells.

Prostaglandin E2 and salbutamol are less effective in raising cAMP levels of alveolar macrophages from ovalbumin-sensitized guinea-pigs as compared to controls

The role of alveolar macrophages in allergic bronchoconstriction is not yet well established but it is clear that these cells possess membrane receptors for the Fc fragments of IgE and may be activated in the inflammatory bronchoalveolar lumen as are mast cells and eosinophils [1]. In experimental models of allergic asthma, based on ovalbumin sensitization, it has been reported that alteration in the number of lung fl-adrenergic receptors may account for the altered response observed in asthma [2]. Since macrophages possess fl-adrenergic receptors and their activity can be modulated by fl-adrenoceptors agonists and drugs increasing intracellular cAMP concentration [3], in this study we compared the in vitro response of alveolar macrophages obtained from control or ovalbuminsensitized guinea-pigs to PGE 2 or salbutamol.