Myeloid dendritic cells induce Th2 responses to inhaled antigen, leading to eosinophilic airway inflammation - PubMed (original) (raw)

Myeloid dendritic cells induce Th2 responses to inhaled antigen, leading to eosinophilic airway inflammation

B N Lambrecht et al. J Clin Invest. 2000 Aug.

Abstract

The aim of this study was to investigate whether dendritic cells (DCs) can induce sensitization to aeroallergen in a mouse model of allergic asthma. Ovalbumin-pulsed (OVA-pulsed) or unpulsed myeloid DCs that were injected into the airways of naive mice migrated into the mediastinal lymph nodes. When challenged 2 weeks later with an aerosol of OVA, activated CD4 and CD8 lymphocytes, eosinophils, and neutrophils were recruited to the lungs of actively immunized mice. These CD4(+) lymphocytes produced predominantly IL-4 and IL-5 but also IFN-gamma, whereas CD8(+) lymphocytes produced predominantly IFN-gamma. Histological analysis revealed perivascular and peribronchial eosinophilic infiltrates and goblet cell hyperplasia. Studies in IL-4(-/-) and CD28(-/-) mice revealed that production of IL-4 by host cells and provision of costimulation to T cells by DCs were critical for inducing the response. Lung CD4(+) T cells strongly expressed the Th2 marker T1/ST2, and signaling through this molecule via a ligand expressed on DCs was essential for the establishment of airway eosinophilia. These data demonstrate that DCs in the airways induce sensitization to inhaled antigen and that molecules expressed on the surface of these cells are critical for the development of Th2-dependent airway eosinophilia.

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Figures

Figure 1

Figure 1

(a) Localization of CFSE+ DC 36 hours after intratracheal injection of 1 × 106 DCs into naive mice. DC were identified in BALF, digested lung tissue, and mediastinal and inguinal LNs as CD3–B220– cells of low autofluorescence, strongly expressing CD11c. Injected DCs can be discriminated from endogenous DCs by green fluorescence of CFSE. The percentage of CFSE+ DCs among all LN cells is indicated. (b) Kinetics of appearance of CFSE+ DC in BALF (open squares, left y axis) and mediastinal LNs (filled squares, right y axis). Representative of five mice per time point in the group.

Figure 2

Figure 2

Effect of OVA exposure on the cellular composition of BALF in animals immunized with DCs. On day 0, mice were immunized by intratracheal administration of 1 × 106 OVA-DCs or control PBS-DCs. On days 14–20, mice were exposed to 30 minutes of daily OVA or PBS aerosol. At 24 hours after the last aerosol (day 21), BAL was performed. Groups are coded as immunization/aerosol exposure. (a) Differential cell counts based on Giemsa staining. (b) Total T lymphocytes (CD3) and subsets (CD4 and CD8) in BALF as determined by flow cytometry. Results are expressed as means ± SEM from eight to ten mice per group. Mono, monocytes; neutro, neutrophils; eosino, eosinophils; lymph, lymphocytes.

Figure 3

Figure 3

Effect of OVA exposure on lung histology in animals immunized with DCs. Mice were treated as in Figure 2. At 24 hours after the last aerosol, lungs were fixed and processed for histological analysis. H&E staining. ×400. (a) Representative section from a mouse immunized with PBS-DC and exposed to PBS aerosol. No abnormalities are noticed. (b and c) Representative sections from mice immunized with OVA-DC and subsequently exposed to OVA-aerosol. These mice develop peribronchial and perivascular eosinophil-rich infiltrates (b) and airway mucosal changes typical of goblet cell hyperplasia (c).

Figure 4

Figure 4

Measurement of cytokine levels in unconcentrated BALF and after in vitro restimulation of LN cells with specific antigen. Mice were treated as in Figure 2. Groups are coded as immunization/exposure. (a) BALF was taken 24 hours after the last aerosol exposure and assayed for the presence of IL-4 and IFN-γ by ELISA. IL-5 could not be detected in BALF. (b and c) Mediastinal and inguinal LNs were resected, and single-cell suspensions were cultured in vitro for 96 hours in the presence of OVA. Results represent means ± SEM from five to eight animals per group.

Figure 5

Figure 5

Intracellular detection of IFN-γ, IL-4, and IL-5 in lymphocytes from BALF of OVA-DC/OVA mice. As described in Methods, cells were stained for IFN-γ (a and b), IL-4 (a), IL-5 (b), and isotype controls (c). Dot plots shown were gated on CD3+CD4+ lymphocytes (top) or CD3+CD8+ lymphocytes (bottom). Number of cells staining for each cytokine are expressed as a percentage of CD4+ or CD8+ cells. Representative of all mice in the group (n = 8).

Figure 6

Figure 6

(a) T1/ST2 expression on BALF cells. Mice were immunized with OVA-DC and subsequently challenged with OVA as described in Methods. Control mice (PBS) were immunized with PBS-DC and challenged with PBS. Single-cell suspensions were surface stained with mAb’s against CD3, CD4, and T1/ST2. Dot plots were gated on CD3+ viable lymphocytes. The percentage of CD4+ lymphocytes expressing T1/ST2+ is indicated. (b) Expression of a putative ligand for T1/ST2 on mouse DCs. Bone marrow DCs were stained for CD11c, MHC class II, and T1/ST2-Ig, followed by secondary anti–hu-Ig-FITC. As a control, staining was performed using hu-Ig as primary antibody (broken line). Histogram was gated on CD11c+ MHC class II+ cells and is representative of three separate experiments.

Figure 7

Figure 7

Effect of blocking the interaction of T1/ST2 with its ligand during sensitization by OVA-DC on the development of eosinophilic inflammation. (a) On day 0 of the experiment, mice received an intratracheal injection of 1 × 106 OVA-DC simultaneously with a rat mAb against T1/ST2 (3E10) or as a control rat IgG. (b) In another experiment, mice received an intratracheal injection of 1 × 106 OVA-DC simultaneously with a T1/ST2-Ig fusion protein or as a control hu-IgG. Mice were subsequently exposed to OVA aerosol from day 14 to day 20 and BALF recovered 24 hours after the last aerosol challenge.

Figure 8

Figure 8

Effect of absence of CD28 on the development of OVA-induced eosinophilic airway inflammation. On day 0, CD28+/+ and CD28–/– mice received 5 × 105 OVA-DC from wild-type mice. Mice were subsequently exposed to OVA aerosol from day 14 to day 20 and BALF was recovered 24 hours after the last aerosol challenge. (a) Differential cell counting on BALF. Results represent means ± SEM from six mice per group. (b) Intracellular staining for cytokines on BALF T cells. Cells were restimulated with PMA/ionomycin in the presence of monensin and stained for IFN-γ and IL-4. Percentage expression of each cytokine on CD3+CD4+ cells is indicated.

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