Cultured lung epithelial cells release antibacterial activity upon contact with Pseudomonas aeruginosa (PA), which is impaired in cystic fibrosis (CF). In order to identify the factors responsible for killing PA by a biochemical approach, we purified antimicrobial activity from supernatants of the A549 lung epithelial cell line, previously stimulated with PA bacteria, by subsequent high performance liquid chromatography. NH(2)-terminal sequencing of a major bactericidal compound revealed it to be identical with human beta-defensin-2 (hBD-2). A mucoid phenotype of PA, but not two nonmucoid PA strains, high concentrations (> 10 microg/ml) of PA lipopolysaccharide, tumor necrosis factor alpha, and interleukin (IL)-1beta, but not IL-6, dose-dependently induced hBD-2 messenger RNA in cultured normal bronchial, tracheal, as well as normal and CF-derived nasal epithelial cells. Genomic analysis of hBD-2 revealed a promoter region containing several putative transcription factor binding sites, including nuclear factor (NF) kappaB, activator protein (AP)-1, AP-2, and NF-IL-6, known to be involved in the regulation of inflammatory responses. Thus, hBD-2 represents a major inducible antimicrobial factor released by airway epithelial cells either on contact with mucoid PA or by endogenously produced primary cytokines. Therefore, it might be important in lung infections caused by mucoid PA, including those seen in patients with CF.

We have investigated the presence of regulated on activation, normal T-cell expressed and probably secreted (RANTES), macrophage inflammatory peptide-1 alpha (MIP-1 alpha), and macrophage chemotactic peptide (MCP-1) in the bronchoalveolar lavage fluid (BALF) obtained from normal (n = 7) and stable asthmatic subjects (n = 8), and studied their kinetic release into asthmatic airways following endobronchial allergen challenge (n = 18). Measurements of RANTES, MIP-1 alpha, and MCP-1 in 10 times (10x) concentrated BALF showed that these three chemokines were present in both normal controls and stable asthmatic patients, but no significant difference between the two groups was found in the levels of the three chemokines. However, at 4 h after allergen challenge, BALF levels of RANTES, MIP-1 alpha, and MCP-1 were significantly increased in fluid obtained from the allergen-challenge site when compared with the saline-challenge control site (median: 175 pg/ml versus 11.5 pg/ml, 258 pg/ml versus 88 pg/ml, and 900 pg/ml versus 450 pg/ml, respectively). At 24 h, levels of the three chemokines returned to baseline values. To investigate whether cells in BALF obtained 4 h after allergen exposure release chemokines, they were cultured for 24 h. BALF cells from the allergen site released more RANTES and MCP-1 than those from the saline site, but released similar amounts of MIP-1 alpha. These findings suggest that RANTES, MIP-1 alpha, and MCP-1 may regulate cell trafficking in asthma in response to allergen exposure.

Eosinophils (Eos) and fibroblasts are known to play a major role in the pathogenesis of bronchial asthma and fibrotic lung disease. Therefore, we investigated whether Th1 and Th2 cytokines stimulate the production of Eo-activating chemokines by lung fibroblasts. Analyses of the culture supernatant using multiple steps of high-performance liquid chromatography demonstrated that interleukin (IL)-4 preferentially stimulates lung fibroblasts to secrete a peak of eosinophil chemotactic activity (ECA) which, upon N-terminal analyses, showed similar sequence to eotaxin, whereas interferon (IFN)-gamma had negligible effect on the release of this chemokine. In contrast, tumor necrosis factor (TNF)-alpha stimulated lung fibroblasts to release two peaks of activity that were found to correspond to eotaxin and regulated on activation, normal T cells expressed and secreted (RANTES), respectively. Interestingly, IL-4 synergized with TNF-alpha to increase greatly the production of three biochemically distinct eotaxin forms. In contrast, IFN-gamma synergized with TNF-alpha to increase RANTES production. Neither IL-2, IL-5, IL-6 nor IL-10 had an effect on lung fibroblasts' capacity to express or release eotaxin and RANTES. Upon appropriate cytokine stimulation, lung fibroblasts were also found to express messenger RNA for monocyte chemotactic protein (MCP)-3 and MCP-4 but not eotaxin-2. However, no ECA like MCP-3 or MCP-4 was detected. These observations suggest that the release of Th1 or Th2 cytokines in the lung tissue polarizes lung fibroblasts to produce either RANTES or eotaxin as major Eo attractants.

Neutrophil infiltration is a major feature in the pathogenesis of the common cold, and respiratory viral infection is the major cause of asthma exacerbations. The factors regulating the neutrophil influx are unknown. Interleukin-8 (IL-8) is a potent neutrophil chemoattractant, which has been implicated in several inflammatory diseases. In this study, we investigated the presence of IL-8 chemokine in the nasal aspirates of asthmatic children (n = 12) in whom asthma was precipitated by proven viral infection. There were increased IL-8 levels in nasal aspirates from children during the virus-induced asthma exacerbations compared with samples from the same children when they had been asymptomatic for 2 wk (medians 863 and < 20 pg/ml, respectively, p < 0.01). Biological relevance was shown in that IL-8 levels correlate with increased nasal aspirate neutrophil myeloperoxidase levels and there was also a correlation between myeloperoxidase levels and upper respiratory symptom severity. Furthermore, we purified IL-8 from these samples, and demonstrated biological neutrophil chemotactic activity. These are the first in vivo data to suggest an important role for IL-8 in neutrophil influx in proven upper respiratory viral infection associated with asthma exacerbations. We suggest that IL-8 might provide a target for therapeutic intervention in virus-induced respiratory diseases.