Release of cytokines by human nasal epithelial cells and peripheral blood mononuclear cells infected with Mycoplasma pneumoniae.

Mycoplasma pneumoniae (Mp) infection is associated with asthma exacerbation in children. We hypothesized that Mp infection may cause airway inflammation by inducing the release of cytokines by respiratory epithelial cells. The levels of chemokines interleukin-8 (IL-8) and released upon activation, normal t cell expressed and secreted (RANTES) released by nasal epithelial cell (NEC) cultures established from asthmatic and nonasthmatic children were measured by ELISA at 4, 24, 48, and 72 hr after cells were inoculated with Mp, and were compared with baseline release of these factors. The presence of MP on apical membranes of NEC after infection was confirmed by transmission electron microscopy, and adherence was shown to be inhibited by erythromycin. Mp infection did not alter NEC release of IL-8 or RANTES at any time point. In contrast, tumor necrosis factor alpha (TNF-alpha) stimulated increased IL-8 at all time points, and respiratory syncytial virus (RSV) infection stimulated RANTES release at 48 and 72 hr by NEC. These results were not significantly different between NEC from asthmatic and nonasthmatic children. As a comparison, peripheral blood mononuclear cells from normal human volunteers were also incubated with Mp and had significantly increased release of IL-2, IL-6, and TNF-alpha. We conclude that Mp, unlike viral pathogens such as RSV, is unlikely to directly stimulate early airway surface cytokine responses via mechanisms involving epithelial cells. We speculate that the chronic presence of mononuclear cells at the airway surface of asthmatics provides a target for Mp-triggered cytokine production.

Effect of broccoli sprouts on nasal response to live attenuated influenza virus in smokers: a randomized, double-blind study.

BACKGROUND: Smokers have increased susceptibility and altered innate host defense responses to influenza virus infection. Broccoli sprouts are a source of the Nrf2 activating agentsulforaphane, and short term ingestion of broccoli sprout homogenates (BSH) has been shown to reduce nasal inflammatory responses to oxidant pollutants. OBJECTIVES: Assess the effects of BSH on nasal cytokines, virus replication, and Nrf2-dependent enzyme expression in smokers and nonsmokers. METHODS: We conducted a randomized, double-blind, placebo-controlled trial comparing the effects of BSH on serially sampled nasal lavage fluid (NLF) cytokines, viral sequence quantity, and Nrf2-dependent enzyme expression in NLF cells and biopsied epithelium. Healthy young adult smokers and nonsmokers ingested BSH or placebo (alfalfa sprout homogenate) for 4 days, designated Days -1, 0, 1, 2. On Day 0 they received standard vaccine dose of live attenuated influenza virus (LAIV) intranasally. Nasal lavage fluids and nasal biopsies were collected serially to assess response to LAIV. RESULTS: In area under curve analyses, post-LAIV IL-6 responses (P = 0.03) and influenza sequences (P = 0.01) were significantly reduced in NLF from BSH-treated smokers, while NAD(P)H: quinoneoxidoreductasein NLF cells was significantly increased. In nonsmokers, a similar trend for reduction in virus quantity with BSH did not reach statistical significance. CONCLUSIONS: In smokers, short term ingestion of broccoli sprout homogenates appears to significantly reduce some virus-induced markers of inflammation, as well as reducing virus quantity. Nutritional antioxidant interventions have promise as a safe, low-cost strategy for reducing influenza risk among smokers and other at risk populations. TRIAL REGISTRATION: ClinicalTrials.gov NCT01269723.

Repeated measurement of nasal lavage fluid chemokines in school-age children with asthma.

BACKGROUND: Inflammatory processes at the mucosal surface may play a role in maintenance of asthma pathophysiology. Cross-sectional studies in asthmatic patients suggest that chemokines such as interleukin 8 (IL-8) are overproduced by respiratory epithelium. OBJECTIVE: To test the hypothesis that chemokine levels are persistently elevated in the respiratory secretions of asthmatic children at a stable baseline. METHODS: We measured nasal lavage fluid (NLF) levels of chemokines and other mediators at 3- to 4-month intervals in a longitudinal study of asthmatic children, with nonasthmatic siblings as controls. RESULTS: In a linear mixed-model analysis, both family and day of visit had significant effects on nasal mediators. Thus, data for 12 asthmatic-nonasthmatic sibling pairs who had 3 or more same-day visits were analyzed separately. For sibling pairs, median eosinophil cationic protein levels derived from serial measurements in NLF were elevated in asthmatic patients compared with nonasthmatic patients, with a near-significant tendency for elevation of total protein and eotaxin levels as well. However, no significant differences were found for IL-8 or several other chemokines. Ratios of IL-13 or IL-5 to interferon-gamma released by house dust mite antigen-stimulated peripheral blood mononuclear cells, tested on a single occasion, were significantly increased for asthmatic patients. CONCLUSIONS: Substantial temporal and family-related variability exists in nasal inflammation in asthmatic children. Although higher levels of eosinophil cationic protein are usually present in NLF of patients with stable asthma compared with patients without asthma, chemokines other than eotaxin are not consistently increased. Eosinophil activation at the mucosal surface is a more consistent predictor of asthmatic symptoms than nonspecific elevation of epithelium-derived inflammatory chemokine levels.

The effect of respiratory synctial virus on chemokine release by differentiated airway epithelium.

Respiratory synctial virus (RSV) infection of undifferentiated airway epithelial cells has been shown to induce the production of chemokines. The purpose of this study was to investigate the vectorial release of interleukin (IL-8) and Released on Activation, Normal T-cell Expressed and Secreted (RANTES) by polarized, well-differentiated respiratory epithelial cells after RSV infection. Human bronchial epithelial cultures were differentiated under air-liquid interface conditions and infected with RSV by the apical or basolateral route. RSV infection was specific to the apical surface. Supernatants were collected at 6 and 48 hours after RSV inoculation, and IL-8 and RANTES were measured by enzyme-linked immunosorbent assay (ELISA). Both IL-8 and RANTES were significantly released by 48 hours following inoculation with RSV. The secretion of each chemokine was greatest after apical inoculation, and secretion was polarized to the basolateral supernatant. Immunohistochemical staining confirmed that RSV infection was specific to ciliated cells, and immunohistochemical staining for chemokines was localized to RSV-infected ciliated cells. The authors conclude that, in differentiated human airway epithelium in vitro, RSV-induced increases in IL-8 and RANTES release are predominantly in the basolateral direction. In epithelial layers, virus-containing cells are the predominant source of the increased chemokine release. The authors speculate that similar processes in vivo influence recruitment of leukocytes to sites of RSV infection.

Bronchoalveolar lavage fluid surfactant protein-A and surfactant protein-D are inversely related to inflammation in early cystic fibrosis.

The pulmonary collectins surfactant protein (SP)-A and SP-D play important roles in innate lung defense, enhancing opsonization of microbes and limiting lung inflammatory responses. To quantify relationships among collectins, bacteria, and inflammation in early cystic fibrosis (CF) airway secretions, bronchoalveolar lavage fluids (BALFs) were collected from children undergoing clinically indicated bronchoscopy. Quantitative bacteriology, differential cell counts, and ELISA for SP-A and SP-D were assessed. Significantly increased numbers of neutrophils relative to bacteria were noted in BALF from CF compared with non-CF subjects. Although SP-A levels tended to be lower in CF compared with non-CF, this was only significant in the presence of bacterial infection. Among CF patients, SP-A concentrations in BALF were inversely related to inflammation, bacterial colony-forming units per milliliter, and age. SP-D levels were significantly decreased in CF patients, and SP-D was rarely detectable in the presence of infection. Among CF patients, SP-D correlated inversely with inflammation and bacterial colony-forming units per milliliter, and there was decreased immunostaining of BALF cells for SP-D in CF. Immunohistochemistry of CF autopsy lung sections for SP-A and SP-D confirmed their paucity at sites of infection and inflammation. We conclude that relative collectin deficiency occurs early in CF airways and is inversely related to inflammation in CF airways.