Myeloid dendritic cells induce Th2 responses to inhaled antigen, leading to eosinophilic airway inflammation (original) (raw)
Injected DCs home to the mediastinal LNs. At various time points after intratracheal injection, we studied the distribution of CFSE-labeled DCs. By 12 hours after instillation, CFSE+DCs could be detected in BALF (14.10 ± 2.99% of cells), digested lung tissue (0.17 ± 0.04% of cells), and draining mediastinal LNs (0.012 ± 0.005%). No CFSE+ DCs could be detected in inguinal LNs. As shown in Figure 1a, by 36 hours DCs had further migrated to the mediastinal LNs (0.07 ± 0.01%) and gradually disappeared from the BALF (5.83 ± 0.88%). The number and kinetics of DC migrating into the draining LNs are shown in Figure 1b. Although by 120 hours, a majority of injected cells had disappeared, some CFSE+ DCs could still be detected in all compartments (1.34 ± 0.15% of BAL; 0.04 ± 0.01% for lung; and 0.016 ± 0.006% for LN).
(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.
Effect of aerosol exposure on airway inflammation in animals immunized with DCs. Analysis on BALF cells revealed that seven OVA aerosol exposures induced a significant increase in total cell counts, mononuclear cells, eosinophils, neutrophils, and lymphocytes in mice that were immunized with 1 × 106 OVA-DCs, compared with animals immunized with PBS-DC (Figure 2a; P < 0.05). Aerosolization with PBS in OVA-DC–immunized mice did not induce significant changes in BALF compared with PBS-DC/PBS–exposed animals or PBS-DC/OVA–exposed animals. Immunization with 1 × 105 OVA-DC and subsequent exposure to OVA aerosol induced an increase in BALF lymphocytes only, whereas immunization with 1 × 104 OVA-DC induced no significant response (data not shown). All subsequent immunizations were therefore performed using 1 × 106 cells.
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.
As studied by flow cytometry and shown in Figure 2b, exposure to OVA aerosol in OVA-DC mice induced a significant increase in both CD4+ and CD8+ T cells compared with PBS exposure. In the absence of secondary OVA exposure, there was no difference in total CD3+ T-cells or T-cell subsets in animals immunized with OVA-DC compared with those immunized with PBS-DC. The early activation marker CD69 was expressed on 62.9 ± 3.2% of CD4+ T cells, and the late activation marker CD25 (IL-2R) was expressed on 34.0 ± 3.5% of CD4+ T cells in the OVA-DC/OVA group compared with 11.5 ± 0.9% and 5.4 ± 0.8%, respectively, in the PBS-DC/PBS group (P < 0.05).
Lung sections from OVA-DC mice exposed to PBS aerosol (data not shown) or PBS-DC mice exposed to OVA (Figure 3a) or PBS aerosol demonstrated no pathological changes. In contrast, histological analysis of lung tissue from OVA-DC/OVA–exposed mice revealed intense multifocal eosinophilic lesions localized mainly in the peribronchial submucosa, but also marked in the perivascular area (Figure 3b). The vascular wall was grossly edematous, and in some inflamed blood vessels, eosinophils were seen to adhere to vascular endothelium. Epithelial changes of goblet cell hyperplasia were readily seen (Figure 3c).
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).
Effect of aerosol exposure on cytokine levels in animals immunized with DCs. The type of Th response being induced by DCs was determined. As shown in Figure 4a, OVA exposure in OVA-DC mice induced a specific twofold increase in the amount of IL-4 detected in BALF, compared with that detected in PBS-DC/PBS mice (P < 0.05). There was no difference in the amount of IFN-γ detectable in BALF. Levels of IL-5 were below the detection limit of our assay.
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.
In vitro restimulation of LN cells (Figure 4, b and c) with OVA demonstrated that mediastinal lymphocytes from OVA-DC/OVA mice produced 27 times more IL-4 (747.4 ± 16.9 vs. 27.2 ± 0.1 pg/mL; P < 0.05), 1.8 times more IL-5 (3,120.2 ± 13.3 vs. 1,698.6 ± 48.6 pg/mL; _P_ < 0.05), and equal amounts of IFN-γ (580 ± 63.6 vs. 876.6 ± 172 pg/mL; _P_ > 0.05) compared with those from PBS-DC/PBS mice. In nondraining LNs, levels of all cytokines were identical between OVA-DC/OVA and PBS-DC/PBS mice.
To investigate whether OVA exposure in OVA-DC mice led to selective infiltration of the lungs with lymphocytes producing either Th1- or Th2-type cytokines, intracellular staining for cytokines was performed on BALF cells (Figure 5). Data on percentage expression on CD4+ and CD8+ cells in OVA-DC/OVA and PBS-DC/PBS mice are summarized in Table 1. Simultaneous staining for IL-5 and IFN-γ was not performed in the PBS-DC/PBS group, due to lower cell yield in this group.
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).
Intracellular staining for cytokines on T cells obtained from BAL
To study the functional role of IL-4 in this model, DCs were grown from wild-type donors in the absence of exogenous IL-4, and transferred into IL-4+/+ or IL-4–/– mice. Aerosolization with OVA after immunization with OVA-DC induced airway eosinophilia in IL-4+/+ mice (47.7 ± 4.75% of BALF cells) but not in IL-4–/– mice (0.2 ± 0.14% of cells). It should be noted that in this experiment, the percentage of BALF eosinophilia in the OVA-DC/OVA group was considerably higher than the 13.6 ± 1.9% in our initial experiments reported in Figure 2a. This can be explained by a prolongation of the OVA-pulsing period, which was initially 2 hours before adoptive transfer and was subsequently prolonged to an overnight pulsing in our later experiments (see Methods). The analysis of BALF of wild-type mice immunized with OVA-DC obtained from IL4–/– or IL4+/+ donors revealed a similar degree of airway eosinophilia in both groups (data not shown).
Role of signaling through T1/ST2. T1/ST2 is a surface marker known to be differentially expressed on Th2 cells (16, 19, 20). The expression of T1/ST2 on CD4+ T cells in BALF and in mediastinal and inguinal LNs was investigated after seven aerosol exposures in OVA-DC or PBS-DC mice. The frequency of T1/ST2-expressing CD4+ cells was highest in BALF (62.0 ± 3.8% in OVA-DC/OVA vs. 8.4 ± 1.5% in PBS-DC/PBS mice (Figure 6a). This decreased to 13.6% of CD4+ T cells in the mediastinal LNs of OVA-DC/OVA mice (versus 3.5% in PBS-DC/PBS mice). In nondraining inguinal LNs, the expression of T1/ST2 was the lowest and similar in both groups (2.9% vs. 2.7% respectively). T1/ST2 was only weakly expressed on CD3+CD4– T cells (i.e., CD8+ T cells). We next determined the expression of a putative ligand for T1/ST2 on DC grown from our bone marrow cultures. As the ligand for murine T1/ST2 has not been cloned, no antibodies that directly stain the T1/ST2 ligand are available. We have previously cloned the extracellular domain of T1/ST2 into a vector containing the human IgG1 constant region, generating an expressed soluble T1/ST2-Ig fusion protein (16). As seen in Figure 6b, DCs bound T1/ST2-Ig weakly but consistently, suggesting the expression of a ligand for T1/ST2.
(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.
We investigated the functional contribution of T1/ST2 to the establishment of eosinophilic airway inflammation by giving either anti-T1/ST2 mAb (3E10) or T1/ST2-Ig during the period of intratracheal injection of DCs. Control animals received either rat IgG or hu-Ig. The intratracheal injection of either 3E10 (Figure 7a) or T1/ST2-Ig (Figure 7b) at the time of sensitization by OVA-DC blocked the development of OVA-induced airway eosinophilia. Moreover, total numbers of monocytes, neutrophils, and lymphocytes were significantly reduced. These changes were also apparent on histological analysis (data not shown).
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.
Role of costimulation through CD28 on T cells. The development of Th2 responses has been shown to depend on strong costimulation provided by CD80/CD86 on APCs to CD28 on T cells (21). DCs were purified from CD28+/+ donors and pulsed overnight with 100 μg/mL of OVA. As expected, immunization with 0.5 × 106 OVA-DC and subsequent exposure to OVA aerosol induced an eosinophilic response in the airways of CD28+/+ mice (32.2 ± 17.7 × 104 eosinophils/lavage) (Figure 8a). However, this eosinophilic response was completely suppressed in CD28–/– mice (1.13 ± 1.1 × 104 eosinophils/lavage). The absence of eosinophilia in CD28–/– mice was also apparent from histological analysis (data not shown). Despite these dramatic differences, the number of CD3+ T cells in BALF was identical between groups (0.6 ± 0.1 × 105 in CD28+/+ versus 0.4 ± 0.2 × 105 in CD28–/–). The number of CD4+ cells expressing CD25 and T1/ST2 was, however, markedly reduced (13.8 ± 1.9% vs. 4.0 ± 1.6% and 14.5 ± 4.6% vs. 2.0 ± 0.4%, respectively) in CD28–/– mice. To determine the type of Th response induced in the absence of CD28, we measured cytokine levels after in vitro restimulation with OVA of T cells from draining LNs of OVA-DC/OVA mice. The levels of IFN-γ and IL-4 were significantly lower in CD28–/– mice (1,050 ± 67.5 pg/mL and 32 ± 3 pg/mL, respectively) compared with CD28+/+ mice (5,441 ± 1,742 pg/mL and 80 ± 44 pg/mL, respectively. The levels of IL-5 were lower in CD28–/– (640 ± 60 pg/mL) than in CD28+/+ mice (877 ± 354 pg/mL), but this did not reach statistical significance. In contrast, intracellular staining for cytokines after polyclonal activation with PMA/ionomycin revealed that individual CD4+ T cells obtained from BALF of OVA-DC/OVA mice produced either IFN-γ or IL-4 in CD28+/+ mice and that the percentage expression of IL-4 in individual cells was not affected by knockout of the CD28 gene (Figure 8b). However, the percentage expression of IFN-γ in CD28–/– T cells was almost twice as high compared with CD28+/+ cells.
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.