We have studied the effect of salmeterol on both P. aeruginosa interactions with the mucosa of nasal turbinate organ cultures and on pyocyanin-induced (20 microg/ml) and elastase-induced (100 microg/ml) damage to nasal epithelial cells. Organ cultures were exposed to salmeterol either by preincubation with 4 x 10(-7) M salmeterol for 30 min or by pipetting 20 microl of 4 x 10(-7) M salmeterol onto the organ culture surface immediately prior to bacterial inoculation. Infected organ cultures (8 h) had significantly (p < or = 0.01) increased epithelial damage, and P. aeruginosa was predominantly associated with damaged epithelium and mucus. Salmeterol significantly (p < or = 0.02) reduced epithelial damage caused by infection and the total number of adherent bacteria (p < or = 0.05), but bacterial distribution on the mucosa was unchanged. Nasal epithelial cells incubated with pyocyanin (20 microg/ml) or elastase (100 microg/ml) for 3 h had significantly (p < or = 0.05) increased cytoplasmic blebbing and mitochondrial damage versus control values. Elastase also significantly (p < or = 0.05) increased cell projection and reduced the level of ciliation. Cells preincubated with salmeterol (2 x 10(-7) M) showed a significant reduction in some features of cell damage caused by both toxins, which was inhibited by the beta2-adrenoceptor antagonist propranolol. Our results indicate that salmeterol reduces P. aeruginosa-induced damage to both organ culture and nasal epithelium.

aaNontypable Haemophilus influenzae is a common commensal bacterium in the upper respiratory tract, present in up to three quarters of normal subjects [1]. However, under permissive conditions it may become pathogenic, and frequently causes upper and lower respiratory tract infections, including otitis media, sinusitis, pneumonia and infective exacerbations of chronic obstructive pulmonary disease. H. influenzae is also commonly isolated from purulent sputum of patients with cystic fibrosis and bronchiectasis [2][3][4].Studies of bacterial interactions with respiratory cells or intact mucosa in vitro have provided important information about the pathogenesis of H. influenzae infections, which may in the future lead to the development of new treatments. Infection causes patchy, and sometimes confluent, damage to epithelium [5,6] and bacteria adhere predominantly to mucus, damaged cells and exposed collagen. H. influenzae endotoxin induces the release of proinflammatory mediators (interleukin (IL)-6, IL-8, tumour necrosis factor (TNF)α and intercellular adhesion molecule (ICAM)-1) from human bronchial epithelial cells [7]. H. influenzae also releases uncharacterized factors which impair mucociliary clearance by stimulating mucus secretion [8], causing ciliary beat slowing and dyskinesia, and damaging epithelial cells [9,10]. Immunoglobulin (Ig)A1 proteases reduce mucosal defence mechanisms [11]. These effects all promote bacterial persistence on the mucosa [12].Salmeterol is a potent β 2 -agonist with a prolonged action [13][14][15]. We have recently shown that salmeterol (4×10 -7 M) reduced the damage that occurred to respiratory epithelium during Pseudomonas aeruginosa infection of nasal turbinate organ cultures, and decreased ultrastructural damage to epithelial cells caused by the P. aeruginosa toxins pyocyanin and elastase [16]. In separate experiments [17,18] it was observed, using light microscopy, that the disruption of epithelial integrity caused by pyocyanin was delayed in the presence of salmeterol (2×10 -7 M) and other agents that increased intracellular cyclic adenosine monophosphate (cAMP). Selective antagonists were used to demonstrate that salmeterol reduced epithelial damage via stimulation of β 2 -adrenoceptors. These data suggest that agents that elevate intracellular cAMP may protect respiratory epithelium from damage caused by bacterial infection.Having established that salmeterol reduces P. aeruginosa-induced epithelial damage of nasal turbinate organ cultures [16], we wished to determine whether this effect was species and tissue specific. We have now investigated the effect of salmeterol on the interaction of H. influenzae with the mucosa of an adenoid organ culture with an air-mucosal interface using scanning electron microscopy. Materials and methods BacteriologyH. influenzae strain SH9 is a nontypable clinical isolate that has been studied previously in our laboratory [6]. SH9 These results suggest that salmeterol protects the respiratory epithelium against Haemophilus influenzae-induced...

The pathogenicity of Haemophilus parainfluenzae (Hpi) in the respiratory tract is unclear, in contrast to the accepted pathogenicity of its close relative non-typable H. influenzae. We have investigated the interaction of two Hpi isolates with the mucosa of adenoid and bronchial tissue organ cultures. The adherence of bacteria to the mucosa of organ cultures, the effect of broth culture filtrates on human nasal epithelium, and interleukin (IL)-8 production by A549 cell cultures was investigated. Hpi 4846 adhered infrequently in clusters of pleomorphic cocco-bacilli to areas of epithelial damage, mucus and unciliated cells in adenoid organ culture experiments at 24 h, but not bronchial mucosa. Hpi 3698 was seen in only one adenoid and no bronchial organ cultures at 24 h. In separate experiments, Hpi 3698 was cleared more rapidly from the centre of the adenoid organ culture and was not cultured at 24 h. Although not adhering to the mucosa at 24 h, Hpi 3698, but not Hpi 4846, caused an increase in the amount of epithelial damage in both types of organ culture. Broth culture filtrates of both strains caused immediate slowing of ciliary beat frequency that progressed, and disrupted epithelial integrity. Dialysed culture filtrates of both strains stimulated IL-8 production by A549 cells, with the culture filtrate of Hpi 3698 being most potent. We conclude that two strains of Hpi varied in their adherence to adenoid tissue, and neither adhered to bronchial tissue. These results lead us to speculate that Hpi is only likely to be a pathogen in the lower respiratory tract when impaired airway defences delay bacterial clearance.