Elsevier

Journal of Immunological Methods

Volume 337, Issue 2, 15 September 2008, Pages 121-131
Journal of Immunological Methods

Research paper
Optimization of methods to study pulmonary dendritic cell migration reveals distinct capacities of DC subsets to acquire soluble versus particulate antigen

https://doi.org/10.1016/j.jim.2008.07.005Get rights and content

Abstract

Dendritic cell migration from the airway to lymph nodes is a key event in the development of airway immunity during infection, allergy, and vaccination. To identify the best approaches to investigate DC migration to lung-draining lymph nodes, we directly compared three methods previously used to track DC migration: airway administration of fluorescent OVA, latex beads, or carboxyfluorescein succinimidyl ester (CFSE). We show that two of the methods employed in optimal conditions—administration of fluorescent OVA or latex particles—have broadly relevant utility in studies of pulmonary DC migration, both in the presence and absence of inflammatory mediators. However, CFSE was of limited value because it induced a robust airway inflammatory response upon instillation. Unexpectedly, antigen-loaded tracers with distinct physical properties differently affected the populations that acquired the tracers and the overall T cell response. Specifically, soluble OVA and OVA formulated as a particulate after conjugation to latex beads were acquired in different proportions in vivo by the two characterized subsets of pulmonary DCs: CD11bhiCD103 and CD11bloCD103+langerin+ DC populations. Consequently, and in line with recent studies that these two subsets of DCs respectively activate CD4+ and CD8+ lymphocyte populations, the physical nature of the antigen delivery vehicle strongly influenced the degree of CD4+ versus CD8+ OVA-specific T cell activation. This finding suggests that changes in the physical presentation of the same antigen delivered to the airway during natural immune responses or vaccinations may markedly affect the character of the T cell response that ensues.

Introduction

Pulmonary dendritic cells (DCs) acquire antigen in the airway and lung and then migrate through lymphatic vessels to the mediastinal lymph node (MLN), where they present antigen and stimulate an immune response (Holt, 1993, Vermaelen and Pauwels, 2003). Because the airway frequently encounters foreign substances, the antigen-presenting role of DCs is critical for the maintenance of pulmonary health. Ideally, harmless antigens induce tolerance, whereas harmful antigens promote effector immune responses (de Heer et al., 2005). One component that influences tolerance versus effector responses is the state of DC maturation (Brimnes et al., 2003). In addition, other cellular mechanisms affect the immunological outcome in response to airway antigens (Hammad and Lambrecht, 2007).

Besides a small number of plasmacytoid DCs, there are two major DC subsets found in the alveolar space and lung parenchyma: CD11c+CD11bloCD103+ DC (here referred to as CD103+ DC or CD11blo DC) and CD11c+CD11bhiCD103 DC (here called CD11bhi DC) (Kirby et al., 2006, Jakubzick et al., 2008). Recent studies are beginning to shed light on their functional roles. Suggesting that the two populations possess markedly distinct functional roles, one group found that CD103+ DCs nearly exclusively promote the proliferation of naïve CD8+ T cells (Belz et al., 2007, del Rio et al., 2007), whereas CD11bhi DC preferentially seem to induce proliferation of CD4+ T cells (del Rio et al., 2007). CD11bhi DCs secrete a substantial number of chemokines during homeostatic and inflammatory conditions, whereas CD103+ DCs mainly secrete chemokines associated with Th2 responses (Beaty et al., 2007). Furthermore, the distribution of these two cell subtypes differs across the pulmonary parenchyma. CD103+ DCs are located along the mucosal lining and vascular wall, while CD11bhi DCs are mainly within perivascular regions (Sung et al., 2006). CD103+ DCs express mRNA for langerin, produce IL-12 upon TLR stimulation, and express tight junction proteins that help them traverse the epithelium and acquire antigen (Sung et al., 2006).

There are three published labeling methods to track DC migration from the lungs to the draining lymph nodes: airway administration of carboxyfluorescein diacetate succinimidyl ester (CFSE), OVA-FITC, or latex particles (Legge and Braciale, 2003, Belz et al., 2004, de Heer et al., 2004, Jakubzick et al., 2006). However, a qualitative and quantitative comparison of these approaches has not been performed. The three methods label DCs in a completely different manner. CFSE labels cells non-selectively, as it spontaneously penetrates cell membranes and irreversibly couples to cellular proteins. OVA-FITC is a soluble protein taken up by pinocytosis, and fluorescent latex particles (0.5μm diameter) are taken up by macropinocytosis or phagocytosis. Here we report on the advantageous and disadvantageous features of each labeling method. In addition, in the course of our study, we uncover a distinct propensity for the two pulmonary DC subsets to acquire soluble versus particulate antigen. Since the two subsets of DCs stimulate CD4+ and CD8+ T cells selectively (Belz et al., 2007, del Rio et al., 2007), we show that skewing antigen delivery to the two types of DCs by altering the physical presentation of the same antigen impacts the relative magnitude of CD4+ versus CD8+ T cell proliferation in response to the antigen.

Section snippets

Mice

C57BL/6 mice were purchased from Jackson Research Laboratories and used for studies at 7–8weeks of age. Mice were housed in a specific pathogen-free environment at Mount Sinai School of Medicine and used in accordance with protocols approved by the Institutional Animal Care and Utilization Committee.

Optimized delivery

We standardized and optimized an intranasal (IN) delivery method to compare all three labeling methods without the need to account for variances in delivery technique. Mice were completely

Relationship between administration of migration tracers and induction of inflammation in the airway and lung

We first analyzed which cells were labeled by CFSE, OVA-FITC, and green fluorescent latex particles by using an optimized intranasal delivery (described in Materials and methods). This method of delivery permits experimental comparison of all three labeling methods while enhancing consistency of delivery among mouse groups. However, whether the delivery was performed as described in the literature (intranasal or intratracheal) or by the optimized IN delivery, the same types of peripheral cells

Discussion

In the literature, there are at least three different methods of labeling pulmonary DCs. These methods may impact experimental outcomes due to selective labeling, induction of cellular death, or inflammation. By making direct comparisons, we were able to provide information about the nuances of each technique that can further guide experimentation.

Besides a small fraction of plasmacytoid DCs, the lung contains two major DC subsets that do not alter their phenotype during migration to the MLN.

Acknowledgments

This work was supported by NIH grant AI49653 to GJR, an institutional training grant to CJ, and a Primary Caregiver Supplement to CJ by NIAID. GJR is an established investigator of the American Heart Association.

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