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In the course of granulocyte-dominated lung inflammation, granulocytes migrate across the endothelium and epithelium of the lung and cause severe tissue damage. To study this process in more detail, we developed a bilayer transmigration model composed of primary human endothelial and lung epithelial cells, simultaneously cultured on opposite sides of Transwell filters. Electron microscopical analysis showed that the morphology of the cells and the expression of junctional proteins remained unaltered and that matrix components were deposited onto the filter. Intriguingly, neutrophil migration was more efficient across the bilayers than across single epithelial monolayers and did not differ from migration across single endothelial monolayers. Coculture experiments showed that endothelial cells stimulated epithelial cells to release IL-6 and that epithelial cells enhanced release of IL-8 from endothelial cells. Together these data reveal bidirectional signaling and enhanced neutrophil migration in a transmigration model of primary human epithelial and endothelial cells.

Infiltration of eosinophils into the lung lumen is a hallmark of allergic asthmatic inflammation. To reach the lung lumen, eosinophils must migrate across the vascular endothelium, through the interstitial matrix, and across the lung epithelium. The regulation of this process is obscure. In this study, we investigated the migration of human eosinophils across confluent monolayers of either human lung H292 epithelial cells or primary human bronchial epithelial cells. Established eosinophil chemoattractants (IL-8, RANTES, platelet-activating factor (PAF), leukotriene B4, and complement fragment 5a (C5a)) or activation of the epithelial cells with IL-1beta induced little eosinophil transmigration (7% in 2 h). In contrast, addition of PAF in combination with C5a induced extensive (20%) transepithelial migration of unprimed and IL-5-primed eosinophils. Eosinophil migration assessed in a Boyden chamber assay, i.e., without an epithelial monolayer, was only slightly increased upon addition of PAF and C5a. Preincubation of eosinophils with the PAF receptor antagonist WEB 2086 only inhibited migration of unprimed eosinophils toward PAF and C5a, whereas preincubation of epithelial cells with WEB 2086 abolished migration of both IL-5-primed and unprimed eosinophils. This latter result indicated the presence of PAF receptors on epithelial cells. Indeed, addition of PAF to epithelial cells induced an increase in cytosolic free Ca2+, which was blocked by the PAF receptor antagonists WEB 2086 and TCV-309. Our results show that PAF induces permissive changes in epithelial cells, and that PAF acts as a chemoattractant and priming agent for the eosinophils.

During allergic disease, leucocytes infiltrate the affected tissues and release their mediators and cytokines. In this way, the local inflammatory process is induced and maintained. Basophilic granulocytes have been demonstrated in lung and sputum of allergic asthmatics, in nasal mucosa and secretion of allergic rhinitis patients, and in skin lesions of atopic dermatitis patients. The number of basophils correlates with the severity of the disease. Analysis of mediator profiles and cellular contents of lavages of nose, skin and lung during allergic late-phase reactions (LPR) have demonstrated histamine, but not tryptase or prostaglandin D2. The histamine-containing cells have been characterized as basophilic granulocytes. This indicates that infiltrating basophils but not mast cells are activated and release their inflammatory contents in the LPR. We are interested in the cellular mechanisms that determine the degranulation of basophils during LPR. Basophil activators, such as allergens and activated complement, are not present at these sites. However, cytokines that prime basophils but do not induce degranulation, such as interleukin-5 (IL-5) and granulocyte/macrophage colony-stimulating factor (GM-CSF), have been detected at sites of LPR. We have now observed that after emptying intracellular Ca2+ stores by means of the Ca2+ adenosine triphosphatase (ATPase) inhibitor, thapsigargin, basophils become extremely sensitive to stimuli that do not affect the Ca2+ stores themselves but that induce degranulation, such as the phorbolester, phorbol myristate acetate (PMA). The most interesting finding was that although both thapsigargin and IL-3, IL-5 or GM-CSF do not induce basophil degranulation by themselves, a 2 min preincubation of basophils with thapsigargin followed by addition of one of these cytokines resulted in extensive histamine release: IL-3 induced 71 +/- 7% histamine release (conc1/2max 6 pM), IL-5 induced 43 +/- 8% histamine release (conc1/2max 41 pM) and GM-CSF induced 57 +/- 10% histamine release (conc1/2max 140 pM). Interestingly, the effect of thapsigargin could be mimicked by platelet-activating factor (PAF) (range 10(-9) to 10(-6) M), although to a lesser extent. Our results indicate that basophil degranulation in tissues during late-phase reactions might be caused by a combination of mediators or cytokines depleting Ca2+ stores, as platelet-activating factor or thapsigargin do, concurrent with activation by interleukin-3, interleukin-5 or granulocyte/macrophage colony-stimulating factor. The response of the basophils towards these cytokines might also be influenced by cell adhesion events, such as binding of basophils via integrins. This is the subject of further study.

For a better insight into the mechanisms determining the recruitment of human basophilic granulocytes from the circulation to sites of allergic reactions, we studied the homotypic aggregation of these cells. The aggregation was studied with95% pure basophil suspensions obtained from peripheral blood in a double-color flow cytometric analysis. Homotypic aggregation was induced by treatment of the basophils with anti-IgE, house dust mite allergen, the chemoattractant FMLP, PMA, or IL-3. The aggregation by anti-IgE was, in part, mediated by interactions with Fc gamma R-II as indicated by 43 +/- 15% inhibition after pretreatment with CD32 antibodies. The aggregation was mediated by beta 2-integrins, as was shown by inhibition of the response by CD18 antibodies. The aggregation induced by anti-IgE, allergen, and PMA displayed comparable kinetics (t1/2 max, 3 to 4 min), in contrast to the degranulation of basophils. FMLP induced the most rapid response (t1/2max, 1.6 min). Inhibition of protein kinase C by staurosporine resulted in a strong (90%) inhibition of the PMA-induced aggregation, whereas the FMLP-induced aggregation was more than doubled (from 11.7 +/- 1.9 to 24.4 +/- 1.9%). Staurosporine did not affect the extent of the anti-IgE-induced aggregation, but it induced a retardation of congruent to 10 min. In most experiments, no clear correlation was found between degranulation and aggregation of human basophils. Most strikingly, IL-3 did not induce degranulation but did induce aggregation. Thus, the homotypic aggregation response of human basophils is induced by intracellular signals not necessarily leading to degranulation. This might be important in the physiologic appearance of basophils at sites of allergic late-phase responses or inflammation.

Salmeterol is a long-acting beta 2-adrenoceptor agonist, with several antiasthma properties. Nimesulide is a nonsteroidal anti-inflammatory drug, supposed to act by inhibition of phosphodiesterase type IV. This might indicate that the effects of both drugs are mediated by an increase in cyclic adenosine monophosphate (cAMP). For salmeterol, it has been shown that it inhibits the influx of eosinophils into the lungs of guinea-pigs after platelet-activating factor (PAF) challenge, suggesting an effect of cAMP on eosinophil migration. For neutrophils, it has been shown that PAF synthesis is inhibited by cAMP. In the present study, we have, therefore, measured the effect of salmeterol and nimesulide on two important human eosinophil functions: chemotaxis; and synthesis of PAF and leukotriene C4 (LTC4). Both drugs were found to be inhibitors of the chemotactic responses of human eosinophils. However, at comparable concentrations, only nimesulide was able to inhibit the synthesis of PAF and LTC4 in activated eosinophils. These results indicate that although the effects of both drugs are thought to be mediated by an increase in cyclic adenosine monophosphate, they have differential effects on eosinophil chemotaxis and synthesis of lipid mediators.