Interactions between T cells and dendritic cells in the airway mucosa precede secondary immune responses to inhaled antigen. The purpose of this study was to identify the anatomical locations where dendritic cell-T cell interactions occur, resulting in T cells activation by dendritic cells. In a mouse model of allergic airway inflammation, we applied whole-mount immunohistology and confocal microscopy to visualize dendritic cells and T cells together with nerves, epithelium, and smooth muscle in three dimensions. Proliferating T cells were identified by the detection of the incorporation of the nucleotide analogue 5-ethynyl-2-deoxyuridine into the DNA. We developed a novel quantification method that enabled the accurate determination of cell-cell contacts in a semiautomated fashion. Dendritic cell-T cell interactions occurred beneath the smooth muscle layer, but not in the epithelium. Approximately 10% of the dendritic cells were contacted by nerves, and up to 4% of T cells formed clusters with these dendritic cells. T cells that were clustered with nerve-contacting dendritic cells proliferated only in the airways of mice with allergic inflammation but not in the airways of negative controls. Taken together, these results suggest that during the secondary immune response, sensory nerves influence dendritic cell-driven T cell activation in the airway mucosa.

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Airway mucosal dendritic cells (DCs), located beneath the epithelium of the conducting airways, are believed to be specialized for immunosurveillance via sampling of antigens from the airway luminal surface. However, the dynamics of airway DC activity has not yet been visualized. We used two-photon microscopy to illuminate the endogenous mucosal DC network in the airways of mice. To characterize DC behavior, we used lung section preparations and an intravital microscopic approach. DCs displayed a heterogeneous movement pattern according to their localization within the airway mucosa: sessile intraepithelial DCs with a dendritiform shape exhibited active probing movements and occasionally formed transepithelial extensions into the airway lumen. In contrast, DCs within the deeper layers of the mucosal tissue migrated fast in an amoeboid manner, without probing movements, and slowed down after aeroallergen challenge. Strikingly, neither of these two mucosal DC populations ingested fluorescently labeled antigens after antigen administration to the airways in the steady state, in contrast to alveolar macrophage/DC populations in the lung periphery. Our results provide a first description of the dynamic behavior of airway mucosal DCs, with their exact role in antigen sampling remaining unclear.

The airway mucosal epithelium is permanently exposed to airborne particles. A network of immune cells patrols at this interface to the environment. The interplay of immune cells is orchestrated by different mediators. In the current study we investigated the impact of neuronal signals on key functions of dendritic cells (DC). Using two-photon microscopic time-lapse analysis of living lung sections from CD11c-EYFP transgenic mice we studied the influence of neuropeptides on airway DC motility. Additionally, using a confocal microscopic approach, the phagocytotic capacity of CD11c+ cells after neuropeptide stimulation was determined. Electrical field stimulation (EFS) leads to an unspecific release of neuropeptides from nerves. After EFS and treatment with the neuropeptides vasoactive intestinal peptide (VIP) or calcitonin gene-related peptide (CGRP), airway DC in living lung slices showed an altered motility. Furthermore, the EFS-mediated effect could partially be blocked by pre-treatment with the receptor antagonist CGRP8–37. Additionally, the phagocytotic capacity of bone marrow-derived and whole lung CD11c+ cells could be inhibited by neuropeptides CGRP, VIP, and Substance P. We then cross-linked these data with the in vivo situation by analyzing DC motility in two different OVA asthma models. Both in the acute and prolonged OVA asthma model altered neuropeptide amounts and DC motility in the airways could be measured. In summary, our data suggest that neuropeptides modulate key features motility and phagocytosis of mouse airway DC. Therefore altered neuropeptide levels in airways during allergic inflammation have impact on regulation of airway immune mechanisms and therefore might contribute to the pathophysiology of asthma.