Lymph node trafficking and antigen presentation by endobronchial eosinophils (original) (raw)
Trafficking of airway eosinophils. To evaluate the capacity of eosinophils within the tracheobronchial lumen to enter tissues, eosinophils recovered from the airways of antigen-sensitized mice after aerosol challenges were fluorescently labeled ex vivo with DiIC16(3) and instilled into the tracheas of both normal and antigen-sensitized mice. Fluorescently labeled eosinophils entered paratracheal lymph nodes within 8 hours (Figures 1 and 2). By 8 hours, labeled eosinophils were visible in the subcapsular region and streaming through the subcapsular sinus. With time the number of eosinophils entering the regional lymph nodes increased, peaking at 24 hours and persisting for at least 120 hours (Figure 2a). By 24 hours after instillation, labeled eosinophils were predominantly located in the T-cell regions of lymph nodes, with only occasional eosinophils in the B-cell areas or the subcapsular sinus (Figure 1). Within 120 hours, fluorescently labeled eosinophils were not detected by microscopy in sections of other examined extrapulmonary lymph nodes, or the spleen, or the liver.
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.)
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.
The migration of eosinophils from the endotracheal lumen into regional lymph nodes was not limited to antigen-elicited airway-derived eosinophils. Peritoneal eosinophils purified from IL-5 C3H/HeN transgenic mice exhibited identical homing to paratracheal lymph nodes when instilled intratracheally into normal C3H/HeN mice (Figure 2a). Because homing of eosinophils within mucosal tissues may be governed by eotaxin expression (18), we assessed whether eosinophil migration from the airways to regional lymph nodes was directed by eotaxin expressed in recipient mice. The migration of eosinophils obtained following aerosol antigen challenge of mice with genetic deletion of the CCR3 receptor for eotaxin (and related chemokines) were examined. _CCR3_–/– eosinophils exhibited identical homing to CCR3+/+ eosinophils from wild-type mice (Figure 2b).
Expression of MHC class II, CD80, and CD86 molecules on airway eosinophils. Human blood eosinophils exhibit low or undetectable expression of class II MHC proteins (7), whereas class II MHC is present on airway eosinophils of asthmatic or after airway antigen challenge in atopic human subjects (14, 15, 21). We determined if murine eosinophils from the airways of antigen-sensitized and -challenged mice expressed molecules involved in the presentation of exogenous antigens. Airway eosinophils recovered from antigen-sensitized and -challenged BALB/c mice expressed high levels of I-Ad (Figure 3a). In contrast, as reported previously (22), peritoneal eosinophils from IL-5 transgenic mice did not express class II MHC proteins (not shown) when isolated, although these eosinophils can be readily induced to express class II proteins in vitro (22). Airway eosinophils, like peritoneal eosinophils from IL-5 transgenic mice (22) (not shown), also expressed high levels of CD80 and CD86 (Figure 3, b and c), two B7 proteins with recognized roles as costimulatory signals for T-cell responses (23).
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-Ad (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.
Eosinophil presentation of airway antigens to sensitized T cells. We assessed the capacity of eosinophils recruited into the airways by aerosol antigen challenge to process and present antigen to which they were exposed within the airways. Splenic T cells from OVA-immunized mice were cultured with increasing numbers of eosinophils purified from the airways of mice challenged with aerosolized OVA (Figure 4). In the absence of eosinophils as APCs, T cells did not proliferate even when incubated with 200 μg/mL of exogenous antigen, confirming the absence of contaminating APCs among the purified T cells. The addition of eosinophils exposed to OVA in vivo yielded significant eosinophil dose-dependent increases in T-cell proliferation, indicative that eosinophils were presenting in vivo–derived and –processed OVA peptides to the sensitized T cells. The addition of exogenous antigen to these eosinophil–T cell cocultures yielded even greater eosinophil dose-dependent proliferation demonstrating that eosinophils were further processing in vitro–derived antigen and serving as APCs.
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. A_P_ < 0.05, in comparison with values for no eosinophils, by ANOVA.
To ascertain that the heightened T-cell proliferation seen with eosinophils exposed in vivo to antigen was not due to a mitogenic or antigen-independent stimulatory process, the antigen specificity of the eosinophil APC function was evaluated. With OVA, BSA, and HGG as independent antigens to elicit antigen-specific T cells and to elicit airway eosinophils by aerosol challenge, T-cell proliferative responses were found only when T cells were cocultured with eosinophils exposed to the homologous antigen (Figure 5, a–c). That eosinophils were the only APCs present in these cultures with purified and antigen-specific T cells was demonstrated by the failure of T cells to proliferate in response to exogenous antigens in the absence of eosinophils (Figure 5, d–f). Again, eosinophils exhibited antigen-specific APC function in response to exogenous antigen added during T-cell proliferation assays, as well as their capacity to process and present antigen to which they were exposed in vivo within the airways.
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.
To evaluate whether eosinophils were providing requisite B7 costimulatory signals for their APC function (23), we assessed the roles of CD80 and CD86 as costimulatory signals in eosinophil antigen presentation in vitro to OVA-sensitized T cells. We cultured 2 × 105 OVA-sensitized T cells and 5 × 104 OVA-exposed eosinophils without exogenous OVA in the presence or absence of inhibitory concentrations of anti-CD80 mAb, anti-CD86 mAb, a combination of both, or CTLA-4Ig. Anti-CD80 and anti-CD86 mAb each partially blocked proliferation, causing 43.9 ± 2.8% and 51.4 ± 4.6% inhibition (mean ± SEM, n = 3), respectively. Both anti-CD80 and anti-CD86 blocking mAbs in combination, or CTLA-4Ig, yielded even greater inhibition of eosinophil-elicited T-cell proliferation (84.7 ± 2.7% and 90.0 ± 1.9%, respectively).
Antigen presentation by airway eosinophils to primed T cells in vivo. To test the APC function of antigen-exposed eosinophils in vivo different numbers of eosinophils from either OVA-, BSA-, or HGG-sensitized and -challenged mice were instilled into tracheas of either OVA-, BSA-, or HGG-sensitized mice. At specific times thereafter, paratracheal lymph nodes were taken and tested for in vivo proliferation by being pulsed immediately with 3H-thymidine and incubated for 16–18 hours. In OVA-sensitized mice that received 5 × 105 OVA-exposed eosinophils, in vivo T-cell proliferative responses in the paratracheal nodes developed within 1 day, peaked at day 3, and then declined over 1 week (Figure 6a). The in vivo T-cell proliferative responses increased with increasing numbers of OVA-exposed eosinophils instilled into the tracheas (Figure 6b). Like the in vitro experiments, eosinophil-induced in vivo T-cell proliferation was also antigen specific, because eosinophils from OVA-sensitized and -challenged mice could only induce proliferation responses of T cells from OVA-sensitized mice, but not from BSA- or HGG-sensitized mice, and the same results were observed with BSA- or HGG-exposed airway eosinophils (Figure 7).
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. A_P_ < 0.01, compared with controls in which no eosinophils were used, by ANOVA.
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.
To delineate the lymph node T-cell subsets induced to proliferate in vivo by the instilled airway eosinophils from antigen-sensitized and aerosol-challenged mice, replicating T cells were labeled in vivo by BrdU incorporation. Lymph node T cells were double-stained with FITC-conjugated anti-BrdU mAb and PE-conjugated anti-CD4 or anti-CD8 mAbs and analyzed by flow cytometry. Only low percentages of BrdU+ CD4+ and BrdU+ CD8+ cells were present in paratracheal lymph nodes of OVA-sensitized mice not given intratracheal eosinophil instillations (Figure 8, a and c). In contrast, the endotracheal instillation of OVA-exposed eosinophils led to significant increases in BrdU+ CD4+ (Figure 8b) but not in BrdU+ CD8+ cells (Figure 8d). Percentages (mean ± SEM) of BrdU+ CD4+ cells in sensitized mice receiving eosinophil instillation (7.7 ± 0.5%) were much higher than that in control mice (2.0 ± 0.1%; n = 5; P = 0.0003, paired t test). Similar results were found with BSA-immunized mice receiving BSA-exposed eosinophils and HGG-immunized mice receiving HGG-exposed eosinophils (not shown). As expected, the endotracheal instillation of fixed, nonviable eosinophils induced no changes in percentages of either BrdU+ CD4+ and BrdU+ CD8+ cells (not shown).
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.
Having established the APC functional activity of airway eosinophils in vivo, we then analyzed the role of B7 costimulatory molecules (23) in in vivo eosinophil antigen presentation to T cells by blocking CD80, CD86, or both. Treatment of OVA-sensitized mice with either anti-CD80 or anti-CD86 mAb each partially suppressed the peak day-3 T-cell proliferation elicited in vivo by tracheal instillation of OVA-exposed eosinophils (42.0 ± 1.7% and 54.4 ± 6.2%; mean inhibitions ± SEM, n = 3, respectively). Treatments with either the combination of anti-CD80 and anti-CD86 mAbs or CTLA-4Ig fusion protein to block both B7-1- and B7-2-mediated costimulation (23) each resulted in about 90% inhibition of in vivo eosinophil-elicited T-cell proliferation (90.1 ± 2.2% and 90.2 ± 2.2%, respectively; mean inhibitions ± SEM, n = 3).