Lymph node trafficking and antigen presentation by endobronchial eosinophils

Abstract

Because eosinophils recruited into the airways in allergic diseases are exposed to inhaled allergens, we evaluated whether eosinophils within the endobronchial lumen can function in vivo as antigen-presenting cells for inhaled antigens. We recovered eosinophils from the airways after aerosol antigen challenge in sensitized mice or from the peritoneal cavities of IL-5 transgenic mice and fluorescently labeled these cells ex vivo. These labeled cells, instilled intratracheally into normal mice, migrated into draining paratracheal lymph nodes and localized to T cell-rich paracortical areas. The homing of airway eosinophils to lymph nodes was not governed by eotaxin, because CCR3(-/-) and CCR3(+/+) eosinophils migrated identically. Airway eosinophils, recovered after inhalational antigen challenge in sensitized mice, expressed MHC class II and costimulatory CD80 and CD86 proteins and functioned in vitro as CD80- and CD86-dependent, antigen-specific, antigen-presenting cells. Moreover, when instilled into the airways of antigen-sensitized recipient mice, airway eosinophils recovered after inhalational antigen challenge stimulated antigen-specific CD4(+) T cell proliferation within paratracheal lymph nodes. Thus, eosinophils within the lumina of airways can process inhaled antigens, traffic to regional lymph nodes, and function in vivo as antigen-presenting cells to stimulate responses of CD4(+) T cells.

Figure 1

Eosinophil migration from the airways into paratracheal lymph nodes. Eosinophils (5 × 10⁵) from the airways of antigen-sensitized and aerosol-challenged mice and labeled with DiIC₁₆(3) (red), were instilled into the tracheas of recipient mice. Paratracheal lymph nodes were harvested at the indicated times after eosinophil instillation. Cryosectioned lymph nodes were stained with HOECHST 33342 (blue) to highlight nuclei and examined by fluorescence microscopy. T cell–rich regions were identified by immunoperoxidase staining with anti-CD3 mAb (brown). Labeled eosinophils were visible in the marginal sinus and the subcapsular region at 8 hours after instillation (a) and started to stream through the subcapsular sinus moving into the paracortical area at 16 hours (b). By 24 hours labeled eosinophils were predominantly located in the T-cell area, whereas few eosinophils were located in B-cell areas and in the subcapsular sinus (c, d). c and d are images of the same section for comparison. (Originally ×250.)

Figure 2

Eosinophil localization within paratracheal lymph nodes. After DiIC₁₆(3)-labeled eosinophils (5 × 10⁵) were instilled into the tracheas of recipient mice, paratracheal lymph nodes were examined by fluorescence microscopy to enumerate migrated eosinophils per square millimeter, as described in Methods. (a) Kinetics of eosinophil appearance in lymph nodes for normal BALB/c mice receiving airway eosinophils isolated from OVA-sensitized and aerosol-challenged BALB/c mice (G), for normal C3H/HeN mice receiving peritoneal eosinophils isolated from IL-5 C3H/HeN transgenic mice (J), and for OVA-sensitized BALB/c mice receiving airway eosinophils isolated from OVA-sensitized and aerosol-challenged BALB/c mice (H). Each result represents the mean ± SEM from 6 mice. (b) Comparisons of the lymph node migration of CCR3^–/– and CCR3^+/+ airway-derived eosinophils isolated from OVA-sensitized and aerosol-challenged mice instilled intratracheally into normal BALB/c mice. Each result represents the mean ± SEM from 3 mice per group.

Figure 3

Expression of MHC class II molecules, CD80 and CD86, on airway eosinophils. Eosinophils, from BAL of BALB/c mice sensitized and aerosol-challenged with antigen, were stained with FITC-conjugated anti-I-A^d (a), anti-CD80 (b), or anti-CD86 (c) mAbs (thick lines) and analyzed by flow cytometry in comparison with isotype control FITC-conjugated IgG (thin lines). Results show a representative experiment from mice sensitized and challenged with OVA. Similar results were found with mice sensitized and aerosol challenged with BSA or HGG.

Figure 4

In vitro stimulation of sensitized T-lymphocyte proliferation by antigen-exposed and antigen-elicited airway eosinophils. OVA-sensitized T cells (2 × 10⁵) were incubated with different numbers of airway eosinophils purified from OVA-sensitized and OVA-challenged mice in the absence and presence of 200 μg/mL exogenous OVA antigen. After 72 hours, cultures were pulsed with ³H-thymidine, and ³H-thymidine incorporation was determined 16–18 hours later. Results are mean ³H-thymidine incorporation ± SD from triplicate cultures. Data from a single experiment are representative of similar findings from 3 other experiments. ^AP < 0.05, in comparison with values for no eosinophils, by ANOVA.

Figure 5

Antigen specificity of in vitro T-cell proliferation induced by antigen-exposed and antigen-elicited airway eosinophils. OVA-exposed (a), BSA-exposed (b), and HGG-exposed (c) airway eosinophils (EOS) were cocultured with nonimmune or OVA-, BSA-, or HGG-sensitized T cells without exogenous antigen. OVA-sensitized (d), BSA-sensitized (e), and HGG-sensitized (f) T cells (TC) were cocultured without eosinophils and exogenous antigen or with eosinophils purified from the airways of OVA-challenged (d), BSA-challenged (e), or HGG-challenged (f) mice with or with 200 μg/mL of exogenous OVA, BSA, or HGG. After 72 hours, cultures of 5 × 10⁴ eosinophils and 2 × 10⁵ T cells were pulsed with ³H-thymidine, and ³H-thymidine incorporation was determined 16–18 hours later. Results are mean ³H-thymidine incorporation ± SD from triplicate cultures. The data from a single experiment are representative of similar findings from 3 other experiments.

Figure 6

Kinetics and dose dependence of in vivo T-cell proliferation elicited by antigen-exposed and antigen-elicited airway eosinophils. (a) OVA-elicited airway eosinophils (5 × 10⁵) were instilled into the tracheas of OVA-sensitized mice, and paratracheal lymph nodes were taken at indicated times and evaluated for in vivo–induced proliferation. (b) Increasing numbers (10⁴–10⁶⁾ of OVA-elicited airway eosinophils were instilled into the tracheas of OVA-immunized mice, and paratracheal lymph nodes, taken 3 days after instillation, were evaluated for in vivo–induced proliferation. In each experiment 3 × 10⁵ lymph node cells were pulsed immediately with ³H-thymidine, incubated for 18 hours, and assessed for ³H-thymidine incorporation. Results are the mean ³H-thymidine incorporation ± SEM of 3 independent experiments. ^AP < 0.01, compared with controls in which no eosinophils were used, by ANOVA.

Figure 7

The antigen specificity of in vivo T-cell proliferation elicited by antigen-exposed and antigen-elicited airway eosinophils. OVA-elicited (a), BSA-elicited (b), and HGG-elicited (c) airway eosinophils (5 × 10⁵) were instilled into the tracheas of nonimmune or OVA-, BSA-, or HGG-immunized mice. Paratracheal lymph nodes, taken 3 days after instillation, were evaluated for in vivo–induced proliferation. Lymph node cells (3 × 10⁵) were pulsed with ³H-thymidine, incubated for 16–18 hours, and assessed for ³H-thymidine incorporation. Results are the mean ³H-thymidine incorporation ± SD of triplicate cultures. The data with a single experiment are representative of similar findings with 3 other experiments.

Figure 8

CD4⁺ but not CD8⁺ T cells respond in vivo to antigen-presenting eosinophils. PBS (a, c) or antigen-exposed eosinophils (b, d) were instilled into the tracheas of antigen-sensitized mice. As described in Methods, mice were administered BrdU, and T cells were purified from lymph nodes 3 days after eosinophil instillation, double-stained with FITC–anti-BrdU mAb and PE–anti-CD4 (a, b) or anti-CD8 mAb (c, d) and analyzed by flow cytometry. Results show 1 experiment representative of 5 independent experiments from OVA-immunized mice receiving OVA-exposed eosinophils; similar results were also found in BSA-immunized mice receiving BSA-exposed eosinophils and HGG-immunized mice receiving HGG-exposed eosinophils. Numbers specify the percentage of cells in each of the 4 quadrants.