Deborah H. Strickland

Regulation of immunological homeostasis in the respiratory tract

The respiratory tract has an approximate surface area of 70 m2 in adult humans, which is in virtually direct contact with the outside environment. It contains a uniquely rich vascular bed containing a large pool of marginated T cells, and harbours a layer of single-cell-thick epithelial tissue through which re-oxygenation of blood must occur uninterrupted for survival. It is therefore not surprising that the respiratory tract is never more than a short step away from disaster. We have only a partial understanding of how immunological homeostasis is maintained in these tissues, but it is becoming clear that the immune system has evolved a range of specific mechanisms to deal with the unique problems encountered in this specialized microenvironment.

Anatomical location determines the distribution and function of dendritic cells and other APCs in the respiratory tract

APCs, including dendritic cells (DC), are central to Ag surveillance in the respiratory tract (RT). Research in this area is dominated by mouse studies on purportedly representative RT-APC populations derived from whole-lung digests, comprising mainly parenchymal tissue. Our recent rat studies identified major functional differences between DC populations from airway mucosal vs parenchymal tissue, thus seriously questioning the validity of this approach. We addressed this issue for the first time in the mouse by separately characterizing RT-APC populations from these two different RT compartments. CD11chigh myeloid DC (mDC) and B cells were common to both locations, whereas a short-lived CD11cneg mDC was unique to airway mucosa and long-lived CD11chigh macrophage and rapid-turnover multipotential precursor populations were predominantly confined to the lung parenchyma. Airway mucosal mDC were more endocytic and presented peptide to naive CD4+ T cells more efficiently than their lung counterparts. However, mDC from neither site could present whole protein without further maturation in vitro, or following trafficking to lymph nodes in vivo, indicating a novel mechanism whereby RT-DC function is regulated at the level of protein processing but not peptide loading for naive T cell activation.

Bidirectional Interactions between Antigen-bearing Respiratory Tract Dendritic Cells (DCs) and T Cells Precede the Late Phase Reaction in Experimental Asthma: DC Activation Occurs in the Airway Mucosa but Not in the Lung Parenchyma

The airway mucosal response to allergen in asthma involves influx of activated T helper type 2 cells and eosinophils, transient airflow obstruction, and airways hyperresponsiveness (AHR). The mechanism(s) underlying transient T cell activation during this inflammatory response is unclear. We present evidence that this response is regulated via bidirectional interactions between airway mucosal dendritic cells (AMDC) and T memory cells. After aerosol challenge, resident AMDC acquire antigen and rapidly mature into potent antigen-presenting cells (APCs) after cognate interactions with T memory cells. This process is restricted to dendritic cells (DCs) in the mucosae of the conducting airways, and is not seen in peripheral lung. Within 24 h, antigen-bearing mature DCs disappear from the airway wall, leaving in their wake activated interleukin 2R+ T cells and AHR. Antigen-bearing activated DCs appear in regional lymph nodes at 24 h, suggesting onward migration from the airway. Transient up-regulation of CD86 on AMDC accompanies this process, which can be reproduced by coculture of resting AMDC with T memory cells plus antigen. The APC activity of AMDC can be partially inhibited by anti-CD86, suggesting that CD86 may play an active role in this process and/or is a surrogate for other relevant costimulators. These findings provide a plausible model for local T cell activation at the lesional site in asthma, and for the transient nature of this inflammatory response.

Reversal of airway hyperresponsiveness by induction of airway mucosal CD4 + CD25 + regulatory T cells

An important feature of atopic asthma is the T cell–driven late phase reaction involving transient bronchoconstriction followed by development of airways hyperresponsiveness (AHR). Using a unique rat asthma model we recently showed that the onset and duration of the aeroallergen-induced airway mucosal T cell activation response in sensitized rats is determined by the kinetics of functional maturation of resident airway mucosal dendritic cells (AMDCs) mediated by cognate interactions with CD4+ T helper memory cells. The study below extends these investigations to chronic aeroallergen exposure. We demonstrate that prevention of ensuing cycles of T cell activation and resultant AHR during chronic exposure of sensitized rats to allergen aerosols is mediated by CD4+CD25+Foxp3+LAG3+ CTLA+CD45RC+ T cells which appear in the airway mucosa and regional lymph nodes within 24 h of initiation of exposure, and inhibit subsequent Th-mediated upregulation of AMDC functions. These cells exhibit potent regulatory T (T reg) cell activity in both in vivo and ex vivo assay systems. The maintenance of protective T reg activity is absolutely dependent on continuing allergen stimulation, as interruption of exposure leads to waning of T reg activity and reemergence of sensitivity to aeroallergen exposure manifesting as AMDC/T cell upregulation and resurgence of T helper 2 cytokine expression, airways eosinophilia, and AHR.

Accelerated antigen sampling and transport by airway mucosal dendritic cells following inhalation of a bacterial stimulus.

An increase in the tempo of local dendritic cell (DC)-mediated immune surveillance is a recognized feature of the response to acute inflammation at airway mucosal surfaces, and transient up-regulation of the APC functions of these DC preceding their emigration to regional lymph nodes has recently been identified as an important trigger for T cell-mediated airway tissue damage in diseases such as asthma. In this study, using a rat model, we demonstrate that the kinetics of the airway mucosal DC (AMDC) response to challenge with heat-killed bacteria is considerably more rapid and as a consequence more effectively compartmentalized than that in recall responses to soluble Ag. Notably, Ag-bearing AMDC expressing full APC activity reach regional lymph nodes within 30 min of cessation of microbial exposure, and in contrast to recall responses to nonpathogenic Ags, there is no evidence of local expression of APC activity within the airway mucosa preceding DC emigration. We additionally demonstrate that, analogous to that reported in the gut, a subset of airway intraepithelial DC extend their processes into the airway lumen. This function is constitutively expressed within the AMDC population, providing a mechanism for continuous immune surveillance of the airway luminal surface in the absence of “danger” signals.

Regulation of Dendritic Cell Recruitment into Resting and Inflamed Airway Epithelium: Use of Alternative Chemokine Receptors as a Function of Inducing Stimulus

Dendritic cells (DC) were purified by flow cytometry from rat tracheal mucosa; they exhibited the phenotypic characteristics of immature DC including high endocytic activity, low CD80/86 expression, and in vitro responsiveness to a broad range of CC chemokines. Daily treatment of adult rats with the selective CCR1 and CCR5 antagonist Met-RANTES reduced baseline numbers of tracheal intraepithelial DC by 50–60%, and pretreatment of animals with Met-RANTES before inhalation of aerosol containing heat-killed bacteria abolished the rapid DC influx into the epithelium that occurred in untreated controls, implicating CCR1 and CCR5 and their ligands in recruitment of immature DC precursors into resting airway tissues and during acute bacterial-induced inflammation. Comparable levels of DC recruitment were observed during airway mucosal Sendai virus infection and after aerosol challenge of sensitized animals with the soluble recall Ag OVA. However, Met-RANTES did not affect these latter responses, indicating the use of alternative chemokine receptors/ligands for DC recruitment, or possibly attraction of different DC subsets, depending on the nature of the eliciting stimulus.

Regulation of T-cell activation in the lung: alveolar macrophages induce reversible T-cell anergy in vitro associated with inhibition of interleukin-2 receptor signal transduction

Alveolar macrophages (AM) are recognized as archetypal ‘activated’ macrophages with respect to their capacity to suppress T‐cell responses to antigen or mitogen, and this function has been ascribed an important role in the maintenance of local immunological homeostasis at the delicate blood : air interface. The present study demonstrates that this suppression involves a unique form of T‐cell anergy, in which ‘AM‐suppressed’ T cells proceed normally through virtually all phases of the activation sequence including Ca2+ flux, T‐cell receptor (TCR) modulation, cytokine [including interleukin‐2 (IL‐2)] secretion and IL‐2 receptor (IL‐2R) expression. However, the ‘suppressed’ T cells fail to up‐regulate CD2, and do not re‐express normal levels of TCR‐associated molecules after initial down‐modulation; moreover, they are unable to transduce IL‐2 signals leading to phosphorylation of IL‐2R‐associated proteins, and remained locked in G0/G1. The induction of this form of anergy is blocked by an NO‐synthase inhibitor, and is reversible upon removal of AM from the T cells, which then proliferate in the absence of further stimulation. We hypothesize that this mechanism provides the means to limit the magnitude of local immune responses in this fragile tissue microenvironment, while preserving the capacity for generation of immunological memory against locally encountered antigens via clonal expansion of activated T cells after their subsequent migration to regional lymphoid organs. In an accompanying paper, we demonstrate that a significant proportion of T cells freshly isolated from lung exhibit a comparable surface phenotype.

Interactions between innate and adaptive immunity in asthma pathogenesis: New perspectives from studies on acute exacerbations

Asthma is a complex multigenic disease. The most frequently encountered form is atopic asthma, which is at its highest prevalence during childhood/young adulthood, and this represents the main focus of this review. The primary risk factor for atopic asthma is sensitization to perennial aeroallergens resulting from a failure to generate protective immunologic tolerance. This tolerance process is orchestrated by airway mucosal dendritic cells and normally results in programming of regulatory T cells, which inhibit activation of the T(H)2 memory cells that, among other activities, drive IgE production and prime the effector populations responsible for IgE-mediated tissue damage. Emerging evidence highlights the complexity of this process, in particular the iterative nature of the underlying interactions between innate and adaptive immune mechanisms in which virtually every signal emanating from one cellular compartment provokes an answering response from the other. To further complicate this picture, the local mesenchyme can also interpose signals to fine tune immune responses to optimally meet local microenvironmental needs. Perturbation of the balance between these interlinked innate and adaptive immune pathways is increasingly believed to be the basis for disease expression, and in the specific case of atopic asthma, the prototypic example of this (discussed below) is acute exacerbations triggered by viral infections.

Size-Dependent Uptake of Particles by Pulmonary Antigen-Presenting Cell Populations and Trafficking to Regional Lymph Nodes

The respiratory tract is an attractive target organ for novel diagnostic and therapeutic applications with nano-sized carriers, but their immune effects and interactions with key resident antigen-presenting cells (APCs) such as dendritic cells (DCs) and alveolar macrophages (AMs) in different anatomical compartments remain poorly understood. Polystyrene particles ranging from 20 nm to 1,000 nm were instilled intranasally in BALB/c mice, and their interactions with APC populations in airways, lung parenchyma, and lung-draining lymph nodes (LDLNs) were examined after 2 and 24 hours by flow cytometry and confocal microscopy. In the main conducting airways and lung parenchyma, DC subpopulations preferentially captured 20-nm particles, compared with 1,000-nm particles that were transported to the LDLNs by migratory CD11blow DCs and that were observed in close proximity to CD3⁺ T cells. Generally, the uptake of particles increased the expression of CD40 and CD86 in all DC populations, independent of particle size, whereas 20-nm particles induced enhanced antigen presentation to CD4⁺ T cells in LDLNs in vivo. Despite measurable uptake by DCs, the majority of particles were taken up by AMs, irrespective of size. Confocal microscopy and FACS analysis showed few particles in the main conducting airways, but a homogeneous distribution of all particle sizes was evident in the lung parenchyma, mostly confined to AMs. Particulate size as a key parameter determining uptake and trafficking therefore determines the fate of inhaled particulates, and this may have important consequences in the development of novel carriers for pulmonary diagnostic or therapeutic applications.

Allergic airways disease develops after an increase in allergen capture and processing in the airway mucosa

Airway mucosal dendritic cells (AMDC) and other airway APCs continuously sample inhaled Ags and regulate the nature of any resulting T cell-mediated immune response. Although immunity develops to harmful pathogens, tolerance arises to nonpathogenic Ags in healthy individuals. This homeostasis is thought to be disrupted in allergic respiratory disorders such as allergic asthma, such that a potentially damaging Th2-biased, CD4+ T cell-mediated inflammatory response develops against intrinsically nonpathogenic allergens. Using a mouse model of experimental allergic airways disease (EAAD), we have investigated the functional changes occurring in AMDC and other airway APC populations during disease onset. Onset of EAAD was characterized by early and transient activation of airway CD4+ T cells coinciding with up-regulation of CD40 expression exclusively on CD11b− AMDC. Concurrent enhanced allergen uptake and processing occurred within all airway APC populations, including B cells, macrophages, and both CD11b+ and CD11b− AMDC subsets. Immune serum transfer into naive animals recapitulated the enhanced allergen uptake observed in airway APC populations and mediated activation of naive allergen-specific, airway CD4+ T cells following inhaled allergen challenge. These data suggest that the onset of EAAD is initiated by enhanced allergen capture and processing by a number of airway APC populations and that allergen-specific Igs play a role in the conversion of normally quiescent AMDC subsets into those capable of inducing airway CD4+ T cell activation.