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Background: Airway dendritic cells (DC) are critical mediators of lung inflammation in asthma, but the characteristics of DC in the airways of healthy children, and children with asthma, are currently unknown., Objective: We sought to identify changes in DC subset distribution and activation profile in paediatric asthma using flow cytometry to analyse induced sputum samples obtained from healthy and asthmatic children., Methods: Lung function and atopic status were determined by spirometry and skin prick testing. Induced sputum samples were analysed using 7-colour flow cytometry to identify airway DC populations (lineage(-) HLA-DR(+) sputum cells expressing either CD11c as conventional DC or CD123 as plasmacytoid DC)., Results: Sputum samples containing lower airway plugs were obtained from 10 healthy children and 8 children with asthma. Lineage(-) HLA-DR(+) DC were successfully identified in all samples, and DC comprised a significantly higher proportion of sputum cells in children with asthma compared with age-matched healthy controls (1.29% vs. 0.67%, P = 0.02). DC expression of the costimulatory marker CD86 was significantly reduced in asthmatic children (73.4% vs. 59.7%, P = 0.04). Sputum DC also included numerous CD1c(+) cells (mean 57% of the total DC population) and low frequencies of cells expressing the subset markers CD141 or CD123, although the proportions of these did not differ between groups., Conclusions: Airway DC can be identified and characterized non-invasively using flow cytometry to analyse paediatric sputum samples. Our data reveal that children with steroid-treated asthma exhibit increased frequency of airway DC with reduced expression of the costimulatory marker CD86, suggesting altered trafficking and/or maturation of these cells either due to asthma or steroid therapies., (© 2014 John Wiley & Sons Ltd.)

Background: Chemical haptens induce both contact and allergic respiratory disease with dendritic cells (DCs) controlling and directing immune responses in vivo. Contact and respiratory haptens may promote differential cytokine production yet distinguishing these effects in vitro remains difficult due to human donor variability. Objective We sought to determine the effect of atopic status on the ability of DC to respond to contact and respiratory sensitizer treatment in vitro as DC from atopic donors are believed to promote Th2-type responses., Methods: Enriched DC from control or atopic donors were treated for 4 h with levels of the contact sensitizer 2,4-dinitrochlorobenzene (DNCB) or the respiratory sensitizer trimellitic anhydride (TMA) that did not reduce cell viability. A sensitive intracellular detection technique was used to measure cytokine production, while T cell responses were assessed in a mixed leucocyte reaction., Results: DC from control, non-atopic, donors produced cytokines differentially in response to sensitizer treatment; DNCB treatment significantly increased the production of Th1 cytokines IL-12 and IFN-gamma while TMA induced the production of IL-13. Control donor DC treated with TMA stimulated less in a mixed leucocyte reaction than untreated cells with any response reduced further by blocking IL-13 in culture. However, DC from atopic donors showed no significant alteration in either cytokine production or T cell stimulatory capacity after sensitizer treatment., Conclusion: Haptens modulate DC by changing the production of cytokines that may play a role in T cell stimulation and subsequent polarization of the immune response. DC from atopic donors were unresponsive to chemical sensitizer treatment, and may be deficient in inducing divergent T cell responses.

Background: Dendritic cells (DC) mediate inflammation in rodent models of allergic airway disease, but the role played by human respiratory-tract DC (hRTDC) in atopic asthma remains poorly defined. Recent data suggest that CD1 antigen presentation by hRTDC may contribute to asthma pathogenesis., Objective: To investigate the influence of hRTDC on the balance between atopy and allergic asthma in human subjects and to determine whether CD1 expression by hRTDC is modulated during asthmatic inflammation., Methods: Sputum cells were induced from steroid-naïve, allergen-challenged and allergen-naïve subjects (atopic asthmatics, atopic non-asthmatics and non-atopic controls). hRTDC were identified using monoclonal antibody labelling and analysis by flow cytometry., Results: hRTDC stained HLA-DR(+) (negative for markers of other cell lineages) were predominantly myeloid and comprised approximately 0.5% of viable sputum cells. Sputum cells were potent stimulators of allogeneic CD4(+) naïve T cells and enrichment/depletion experiments correlated stimulatory potency with DC numbers. Sputum contained cells that exhibited typical dendritic morphology when analysed by electron microscopy. Myeloid hRTDC were endocytically active, but uptake of FITC-dextran was enhanced in cells from asthmatics (P<0.001). Despite their increased endocytic capacity, asthmatic myeloid hRTDC appeared mature and expressed increased levels of maturation markers (P<0.05-P<0.001), CD1c, CD1d and langerin (P<0.05). CD1c expression by asthmatic myeloid hRTDC was enhanced upon in vivo allergen challenge (three to ninefold within 24 h; P<0.05). CD11c(-)CD123(high) hRTDC were only detected in asthmatic sputum and were increased in number following allergen challenge., Conclusion: Despite limited cell numbers, it proved possible to analyse human RTDC in induced sputum, providing evidence that increased antigen uptake and enhanced CD1 presentation by activated hRTDC may contribute to allergic airway disease. CD1 presentation by hRTDC in atopic asthma may therefore constitute a novel target for future intervention strategies.