The Leukotriene B4 Receptor (BLT1) Is Required for Effector CD8+ T Cell-Mediated, Mast Cell-Dependent Airway Hyperresponsiveness

Christian Taube, Nobuaki Miyahara, Vanessa Ott, Brad Swanson, Katsuyuki Takeda, Joan Loader, Leonard D. Shultz, Andrew M. Tager, Andrew D. Luster, Azzeddine Dakhama and Erwin W. Gelfand
J Immunol March 1, 2006, 176 (5) 3157-3164; DOI: https://doi.org/10.4049/jimmunol.176.5.3157

Abstract

Studies in both humans and rodents have suggested that CD8+ T cells contribute to the development of airway hyperresponsiveness (AHR) and that leukotriene B4 (LTB4) is involved in the chemotaxis of effector CD8+ T cells (TEFF) to the lung by virtue of their expression of BLT1, the receptor for LTB4. In the present study, we used a mast cell-CD8-dependent model of AHR to further define the role of BLT1 in CD8+ T cell-mediated AHR. C57BL/6+/+ and CD8-deficient (CD8−/−) mice were passively sensitized with anti-OVA IgE and exposed to OVA via the airways. Following passive sensitization and allergen exposure, C57BL/6+/+ mice developed altered airway function, whereas passively sensitized and allergen-exposed CD8−/− mice failed to do so. CD8−/− mice reconstituted with CD8+ TEFF developed AHR in response to challenge. In contrast, CD8−/− mice reconstituted with BLT1-deficient effector CD8+ T cells did not develop AHR. The induction of increased airway responsiveness following transfer of CD8+ TEFF or in wild-type mice could be blocked by administration of an LTB4 receptor antagonist confirming the role of BLT1 in CD8+ T cell-mediated AHR. Together, these data define the important role for mast cells and the LTB4-BLT1 pathway in the development of CD8+ T cell-mediated allergic responses in the lung.

One hallmark of allergic asthma is the development of airway hyperresponsiveness (AHR)4 (1); T cells and the cytokines they release, especially IL-13, are thought to play a central role in the development of increased airway reactivity (2, 3, 4). The role of CD4+ T lymphocytes in the development of AHR is well-documented (5), but there is now increasing evidence that CD8+ T cells also contribute to the development of increased airway reactivity (6), in light of the findings that Ag-primed CD8+ T cells were shown to be a source of IL-13 (7).

Ag-activated populations of CD8+ T cells can be distinguished by the expression of CD62 ligand (L-selectin, CD62L) and CCR7 and in their functional and migratory properties (8, 9, 10). Ag-experienced CD8+ central memory T cells (TCM) express high levels of CD62L and CCR7 and home preferentially to lymph nodes. In contrast, effector CD8+ T cells (TEFF) express low levels of CD62L and CCR7 and traffic more efficiently to nonlymphoid tissues. These subsets of CD8+ T cells can be differentially cultured in vitro: when cultured in the presence of IL-15, CD8+ T cells acquire the phenotypic and functional characteristics of TCM, whereas activated CD8+ T cells cultured in the presence of IL-2 show characteristics of TEFF (10, 11).

BLT1 is a specific G protein-coupled cell surface receptor for leukotriene B4 (LTB4) (12, 13). LTB4 signaling through BLT1 has been shown to contribute to granulocyte and macrophage accumulation at the sites of inflammation (14, 15). Recent studies have shown that BLT1 is highly expressed on effector phenotype CD8+ T cells generated in vitro (TEFF) (16, 17). This is in contrast to TCM, which express low levels of BLT1. Consistent with these observations, chemotaxis of TEFF, but not TCM, is induced by soluble LTB4 (16, 17) and LTB4 produced by activated mast cells (17).

To investigate the interaction of activated CD8+ T cells and mast cells in vivo, we used a model of passive sensitization of mice with allergen-specific IgE and airway exposure to allergen, in the absence of adjuvant (18). This model of AHR is similar to repeated inhaled allergen exposure which is known to be dependent on mast cells, expression of FcεRI, and IL-13 (19). This mast cell-dependent model, which bypasses the need for host-derived IgE, was used to define the functional properties of memory CD8+ T cells, especially TEFF cells, as well as their dependence on BLT1 expression. We show that transfer of TEFF but not TCM reconstitutes AHR and airway eosinophilia in passively sensitized CD8-deficient mice and these responses were dependent on BLT1 expression on the TEFF. These data identify critical interactions between mast cells, the LTB4-BLT1 pathway, and TEFF in the development of CD8+ T cell-mediated airway reactivity.

Materials and Methods

Animals

Female BALB/cByJ mice, mast cell-deficient mice ((WB/ReJ-kitW/+ × C57BL6J-kitW-v/+)F1-(W/Wv) mice), congenic WBB6F1 wild-type (WT) mice, B6.Cg-Tg(TcrOVA) 1100 mjb (OT-1 mice) expressing a transgenic TCR that is specific for OVA257–264 (SIINFEKL) peptide in C57BL/6 mice (20), B6.129S2-cd8atm1mak (CD8−/−) mice, generated by targeting the CD8α chain gene in C57BL/6 mice (21), or C57BL/6 WT mice from 8 to 12 wk of age were used. Ltb4r1tm1Adl (BLT1−/−) mice F1 hybrids of C57BL/6 and 129Sv/J genetic background (22) were backcrossed into the C57BL/6 genetic background for nine generations. BLT1-deficient OT-1 mice were generated by mating BLT1−/− mice with OT-1 mice. Mice with a disruption of the α subunit of the high-affinity IgE receptor Fcer1atm1Rav (FcεRI−/−) (BALB/c congenic background) (23) (provided by Drs. D. Dombrowicz and J. P. Kinet, Harvard Medical School, Boston, MA), IL-13-deficient IL13tm1.1Anjm (IL-13−/−) mice (24) (BALB/c background) (provided by Stanford University, Stanford, CA) were all bred and maintained in the animal facility at the National Jewish Medical and Research Center. All experimental animals used in this study were under a protocol approved by the Institutional Animal Care and Use Committee of the National Jewish Medical and Research Center.

Experimental protocols

Experimental groups consisted of four mice per group and each experiment was performed at least twice. Mice were injected i.v. on 2 consecutive days with 2 μg of OVA-specific IgE, derived from an anti-OVA Ab secreting hybridoma cell lines (18), in a volume of 200 μl and then exposed to OVA (Grade V; Sigma-Aldrich) via the airways as described previously (18). Briefly, a solution of 1% OVA in 0.9% saline was delivered by ultrasonic nebulization for 20 min on days 3 and 4 following the second OVA-IgE injection. AHR was assessed 48 h after the last nebulization.

In vitro differentiation of TEFF and TCM

In vitro differentiation of TEFF cells and TCM was conducted as previously described (10, 17). Lymph node and spleen mononuclear cells (MNC) collected from BLT1+/+ and BLT1−/− OT-1 TCR transgenic mice were cultured with 1 μM OVA257–264 (SIINFEKL) peptide. Following culture for 2 days, cells were washed and cultured with IL-2 (20 ng/ml) or IL-15 (20 ng/ml). After 7 days, it was determined that the cultures contained >99% αβ/CD8+ T cells. CD4+ cells, CD11c+ cells, NK1.1+ cells, or γδ+ cells were <0.1%. The cells were >90% CD8+ memory cell phenotype, as shown by the CD122high/CD44high cell surface phenotype. TEFF and TCM derived through this protocol showed phenotypic and functional characteristics of effector and central memory CD8+ T cells in vivo (10, 17).

In vitro-differentiated TEFF and TCM were transferred to recipient mice by i.v. injection into the tail vein. Each recipient received 5 × 106 TEFF or TCM cells 2 h before the first OVA challenge.

IL-13 production

To assess IL-13 production, TEFF and TCM obtained from BLT1+/+ OT-1 mice were activated in culture (2 × 105 cells) in 96-well plates precoated for 16 h with 2 μg/ml each of monoclonal anti-CD3 (145.2C11) and anti-CD28 (37.51) for 24 h in a humidified atmosphere of 5% CO2 and 37°C. Cell-free supernatants were harvested and levels of IL-13 were assessed by ELSIA (R&D Systems) according to the manufacturer’s directions. The limits of detection were 4 pg/ml.

Administration of the soluble IL-13Rα2-IgGFc fusion protein (sIL-13Rα2-Fc) and LTB4 receptor antagonist

Murine IL-13Rα2-hIgG fusion protein (25) was obtained from Dr. D. Donaldson (Wyeth, Cambridge, MA). In the passive sensitization protocol, IL-13Rα2-hIgG fusion protein was administered by i.p. injection (300 μg/mouse), 1 h before each OVA challenge. As a control, the same amount of human IgG was injected at the identical time points.

The LTB4 receptor antagonist (CP105, 696, provided by Pfizer Pharmaceuticals) (26, 27) or vehicle was administered by gavage at 50 mg/kg (suspended in 100 μl of hydroxypropylmethylcellulose (Abbott Laboratories) 1 day before starting the OVA challenge and 1 h before each OVA challenge.

Assessment of airway smooth muscle responsiveness

Forty-eight hours following the last airway challenge, AHR was monitored as airway smooth muscle responsiveness to electric field stimulation (EFS), measured as described previously (28). Tracheas were removed, and 0.5-cm preparations were placed in Krebs-Henseleit solution suspended by triangular supports transducing the force of contractions. EFS with an increasing frequency from 0.5 to 30 Hz was applied, and the contractions were measured. The duration of the stimulation was 1 ms. Frequencies resulting in 50% of the maximal contraction (ES50) were calculated from linear plots from each individual animal and were compared between the groups.

Bronchoalveolar lavage (BAL)

Forty-eight hours following the last airway challenge, lungs were lavaged via the tracheal tube with HBSS. Total leukocyte numbers were counted by cell counter (Coulter Counter; Coulter). Cytospin slides were stained with Leukostat (Fisher Diagnostics) and differentiated by standard hemological procedures. For measurement of LTB4, BAL was performed 24 h after the last airway challenge.

Measurement of LTB4 levels in the BAL fluid

LTB4 levels in the BAL fluid were measured by ELISA (R&D Systems). ELISAs were performed according to the manufacturer’s directions. The limits of detection were 5.63 pg/ml.

Lung cell isolation

Lung cells were isolated as previously described using collagenase digestion (18). Cells were resuspended in HBSS and MNC were purified by Ficoll-Hypaque gradient centrifugation.

Flow cytometry

After purification, 1 × 106 cells were incubated with allophycocyanin-conjugated anti-CD3 and FITC-conjugated anti-CD8 Abs (BD Pharmingen), and then analyzed by flow cytometry (FACSCalibur; BD Immunocytometry Systems) as previously described (7). The number of CD8+ T cells in the lung or BAL was derived by multiplying the percentage of stained cells by the total number of lung cells or BAL fluid cells isolated.

Statistical analysis

ANOVA was used to determine the levels of difference between all groups. Comparisons for all pairs were performed by Tukey-Kramer honest significant difference test and p values for significance were set to 0.05. Values for all measurements were expressed as the mean ± SEM.

Results

Development of increased airway reactivity following passive sensitization is dependent on mast cells, CD8 T cells, IL-13, and expression of FcεRI and BLT1

Mast cell-deficient mice, FcεRI−/− mice, CD8−/− mice, and the respective WT control mice were passively sensitized with OVA-specific IgE and then challenged with nebulized OVA on 2 consecutive days. All passively sensitized and challenged WT mice (independent of the strain) showed an increased response to EFS (defined as a decrease in ES50) when compared with the passively sensitized but saline-challenged or nonsensitized but allergen-challenged animals (Figs. 1⇓, A–C). In contrast, mast cell-deficient mice, FcεRI−/− mice, and CD8−/− mice, passively sensitized with OVA-IgE and challenged with OVA, demonstrated EFS responsiveness which was similar to nonsensitized control mice (Fig. 1⇓). These data extend our previous findings (19) and identify an IgE-FcεRI-mast cell-CD8 pathway in the development of altered airway reactivity in this passive sensitization and allergen challenge model.

FIGURE 1.
FIGURE 1.

Development of increased airway reactivity following passive sensitization is dependent on mast cells, IL-13, and expression of FcεRI, CD8, and BLT1. A, Mast cell-deficient mice (W/Wv) (n = 8 in each group). B, FcεRI-deficient mice (FcεRI−/−) (n = 12 in each group) and IL-13-deficient mice (n = 8 in each group). C, CD8-deficient mice (CD8−/−) (n = 12 in each group). D, BLT1-deficient mice (BLT1−/−) (n = 8 in each group) and similar numbers of the respective WT controls (+/+, BALB/c and C57/BL6) were passively sensitized with OVA-IgE (OVA-IgE+) exposed to nebulized OVA (OVA neb+) or saline (OVA neb−), and then response to EFS was determined. E–G, LTB4 levels in BAL fluid of CD8−/−, FcεRI−/−, and mast cell-deficient mice. ∗, p < 0.05 compared with passively sensitized and saline-exposed WT mice.

In a comparable model of mast cell-dependent development of AHR, IL-13 was shown to be essential (19). In the passive sensitization model, development of altered tracheal smooth muscle reactivity was shown to be dependent on IL-13: IL-13-deficient mice failed to develop increased reactivity when passively sensitized and challenged when compared with WT mice (Fig. 1⇑B).

Mice lacking expression of the high-affinity receptor for LTB4 (BLT1) failed to develop AHR following systemic sensitization and challenge (29). BLT1−/− mice similarly showed no increase in airway reactivity following passive sensitization and OVA exposure when compared with WT mice (Fig. 1⇑D).

LTB4 levels in BAL fluid is decreased in FcεRI−/− mice and mast cell-deficient mice

We previously suggested that the failure of mast cell-deficient mice or FcεRI−/− mice to develop AHR in a similar system could be overcome by adoptive transfer of bone marrow-derived mast cells from WT or IL-13−/− mice (19) and that the failure of CD8−/− mice to respond could be overcome by IL-13+/+ but not IL-13−/− CD8 T cells (7). These data suggested that activated mast cells were critical to the recruitment of CD8+ T cells and BLT1-expressing CD8 T cells.

Passive sensitization and OVA challenge led to increased levels of LTB4 in the BAL fluid of C57BL/6 mice as well as in CD8−/− mice (Fig. 1⇑E). This increase was more significant at 24 h than 48 h after the last challenge (data not shown). However, following passive sensitization and challenge, levels of LTB4 in mast cell-deficient and FcεRI−/− mice were significantly lower compared with WT mice (Fig. 1⇑, F and G). These data point to a critical pathway for LTB4 production from mast cells through FcεR1 ligation following stimulation with OVA and OVA-specific IgE.

Transfer of TEFF, but not TCM, induces increased airway reactivity in CD8−/− mice following passive sensitization

As passive sensitization and allergen exposure did not increase airway reactivity in CD8−/− mice, we examined the capacity of allergen-specific CD8+ transferred TEFF and TCM to reconstitute this IgE-mast cell-dependent response. In these experiments, we adoptively transferred in vitro-generated SIINFEKL-specific CD8+ TEFF and TCM cells obtained from BLT1+/+ OT-1 mice. Transfer of TEFF and TCM and OVA challenge into mice which were not passively sensitized did not alter airway reactivity to EFS (Fig. 2⇓A). Transfer of TEFF into passively sensitized OVA-challenged CD8−/− mice increased their response to EFS, comparable to passively sensitized and OVA-exposed WT mice. In contrast, transfer of TCM into similarly sensitized and challenged CD8−/− mice failed to affect the response to EFS (Fig. 2⇓A).

FIGURE 2.
FIGURE 2.

Transfer of TEFF but not TCM to passively sensitized CD8−/− mice increases airway reactivity. In vitro-generated TEFF and TCM obtained from BLT1+/+ OT-1 mice were transferred to nonsensitized (OVA-IgE−) or passively sensitized (OVA-IgE+), C57BL/6 (CD8+/+), or CD8-deficient (CD8−/−) recipients, 2 h before the first saline or OVA exposure. A, Transfer of TEFF or TCM; n = 12 in each group. ∗, p < 0.05 compared with nonsensitized CD8+/+ recipients. #, p < 0.05 compared with all other CD8−/− recipients. B, Representative flow cytometry of transferred TEFF and TCM in the lung. Lung cells were isolated and stained with anti-CD3 and anti-CD8 Ab as described in Materials and Methods. Quadrants were based on isotype control staining. Numbers of CD8+ T cells in the lung (C) and BAL (D) in CD8−/− mice following transfer of TEFF and TCM in the same groups as shown in A. The numbers of cells were calculated as described in Materials and Methods. ∗, Significant differences (p < 0.05) compared with other groups.

Migration of TEFF and TCM

We next determined whether the differences in the response between TEFF and TCM recipients were due to differences in migration to the lung as reported in other models (30). Lungs were examined 48 h following challenge of CD8−/− mice (Fig. 2⇑B). In these CD8−/− mice, transferred cells were easily identified as CD3+/CD8+ cells. Overall, transferred TEFF amounted to 3.5 ± 0.3% (mean ± SEM, n = 8) of total MNC in the lungs following passive sensitization and challenge whereas transferred TCM were found at a lower percentage (1.4 ± 0.3% of total lung MNC). Fig. 2⇑, C and D, summarizes the numbers of transferred TEFF in the lungs and BAL fluid. These data indicate that higher numbers of transferred TEFF were recovered from the lung and BAL compared with TCM. No such effects were observed if reconstituted mice were not passively sensitized with OVA-specific IgE or were not challenged (data not shown).

BLT1+/+ TEFF, but not BLT1−/−, restore airway reactivity in CD8−/− mice

As shown in Fig. 1⇑D, BLT1−/− mice showed no increase in airway reactivity following passive sensitization and OVA challenge when compared with WT mice. To address whether expression of BLT1 on CD8+ TEFF was required for reconstitution of airway reactivity, CD8−/− mice received in vitro-generated BLT1−/− or BLT1+/+ CD8+ TEFF cells. Fig. 3⇓A shows that the CD8−/− mice which received TEFF from BLT1+/+ mice fully restored airway reactivity to levels that were comparable to those seen in WT mice (Fig. 3⇓A), whereas transfer of BLT1−/− TEFF failed to restore airway reactivity. Transfer of BLT1+/+ TEFF into WT mice did not further enhance airway reactivity (data not shown).

FIGURE 3.
FIGURE 3.

Transfer of BLT1+/+ TEFF but not BLT1−/− TEFF to passively sensitized CD8−/− mice can induce increased airway reactivity. In vitro-generated BLT1+/+ TEFF and BLT1−/− TEFF were transferred to nonsensitized or passively sensitized recipient CD8-deficient (CD8−/−) mice 2 h before the first OVA exposure. A, Airway responsiveness. CD8+/+ mice receiving no cells are also shown; n = 8 in each group. #, p < 0.05 compared with challenged only CD8+/+ mice; ∗, p < 0.05 compared with all other CD8−/− recipients. B, Representative flow cytometry of BLT1+/+ and BLT1−/− TEFF in the lung. Lung cells were isolated and stained with anti-CD3 and anti-CD8 Ab as described in Materials and Methods. Quadrants were based on isotype control staining. Numbers of CD8+ TEFF in the lung (C) and BAL (D) in CD8−/− mice following transfer of BLT1+/+ and BLT1−/− TEFF. The numbers of cells were calculated as described in Materials and Methods. Groups are the same as in A. ∗, Significant differences (p < 0.05) compared with other groups.

Migration of BLT1−/− TEFF into the airways is impaired following passive sensitization

The accumulation of adoptively transferred BLT1+/+ or BLT1−/− TEFF in the lungs of CD8−/− mice was determined 48 h after the last challenge. Overall, transferred BLT1+/+ TEFF cells amounted to 3.4 ± 0.3% of total lung MNC following passive sensitization and OVA challenge, whereas transferred BLT1−/− TEFF cells were found at a much lower percentage (0.8 ± 0.2% of total lung MNC) (mean ± SEM, n = 8) (Fig. 3⇑B). Fig. 3⇑, C and D, summarizes the numbers of transferred TEFF detected in the lungs and BAL fluid. Lower numbers of BLT1−/− TEFF were detected in the lung and BAL compared with BLT1+/+ TEFF following passive sensitization and OVA challenge. In the absence of passive sensitization or OVA challenge (data not shown), no increase in lung BLT1+/+ TEFF was observed.

In vitro-generated TEFF produce increased amounts of IL-13 compared with TCM following stimulation

As the development of AHR was shown to be dependent on IL-13 and CD8+ TEFF, we determined whether the differences between TEFF and TCM were due to differences in capacity for IL-13 production. To examine the capacity for IL-13 production, in vitro-generated SIINFEKL transgenic TEFF and TCM obtained from BLT1+/+ OT-1 mice were stimulated in vitro with anti-CD3 and anti-CD28. Twenty-four hours following stimulation, supernatant was collected and assayed for IL-13. Levels of IL-13 were significantly higher in supernatants from cultured TEFF compared with cultured TCM following cross-linking of the surface receptors (Fig. 4⇓).

FIGURE 4.
FIGURE 4.

TEFF produce more IL-13 than TCM following TCR stimulation. In vitro-generated TEFF and TCM obtained from BLT1+/+ OT-1 mice were incubated in medium or stimulated with CD3/CD28 Ab. After 24 h, cell-free supernatants were collected and analyzed for IL-13 levels by ELISA. ∗, p < 0.01 compared with medium alone. #, p < 0.01 compared with medium alone. ∗∗, p < 0.01 comparing stimulated TEFF to stimulated TCM.

Increased airway reactivity following TEFF can be abolished by LTB4 receptor blockade and neutralization of IL-13

TEFF have previously been shown to express the LTB4 receptor 1 (BLT1) and migrate toward LTB4 and activated mast cells (17). We have shown that BLT1 expression on TEFF is essential for development of AHR following systemic sensitization and challenge (29) and that BLT1−/− mice fail to develop altered airway reactivity following passive sensitization and challenge. Cumulatively, these data indicated that CD8+ TEFF were essential to the development of altered airway responsiveness following passive sensitization and airway challenge and that their function is dependent on the expression of BLT1, BLT1 mediating their accumulation in the lung. Further, their ability to reconstitute AHR was, at least in part, linked to their capacity to produce IL-13 (Fig. 4⇑ and Ref.30). To determine whether BLT1 and IL-13 were important in this TEFF-IgE-mast cell-dependent model, we treated passively sensitized CD8−/− mice with an LTB4 receptor antagonist or an inhibitor of IL-13 before the OVA challenges. Treatment with the LTB4 receptor antagonist completely abolished the effect of TEFF obtained from BLT1+/+ OT-1 mice on the development of increased airway reactivity (Fig. 5⇓A) and resulted in a decreased accumulation of TEFF in the lung (Fig. 5⇓B). A similar effect was achieved by inhibition of IL-13 activity with sIL-13Rα2, which similarly abolished the effect of TEFF transfer on AHR in passively sensitized CD8−/− mice (Fig. 5⇓C).

FIGURE 5.
FIGURE 5.

Reconstitution of increased airway reactivity following TEFF transfer to CD8−/− mice is dependent on expression of the LTB4 receptor and IL-13. In vitro-generated TEFF obtained from BLT1+/+ OT-1 mice were transferred 2 h before the first OVA exposure. A, Airway responsiveness in TEFF recipient mice receiving LTB4 receptor antagonist or vehicle; n = 12 for all groups. ∗, p < 0.05 compared with mice following vehicle treatment. B, Lung cell numbers in the same groups as A. ∗, p < 0.05 compared with mice following vehicle treatment. C, Airway responsiveness in TEFF recipient mice receiving sIL-Rα2 or human IgG; n = 8 in each group. ∗, p < 0.05 compared with the human IgG-treated group.

To confirm that these inhibitors were equally effective in intact animals (CD8-sufficient), the inhibitors were administered to C57BL/6 mice after passive sensitization and before challenge. As shown in Fig. 6⇓, treatment with either the LTB4 receptor antagonist or the IL-13 inhibitor prevented the development of altered airway reactivity in these WT mice to the same extent as the BLT1−/− or IL-13−/− mice or the BLT1 and IL-13 targeted CD8−/− mice.

FIGURE 6.
FIGURE 6.

Effect of blocking BLT1 or inhibiting IL-13 in normal (WT) mice. A, Airway responsiveness in WT mice treated with the LTB4 Rc receptor antagonist or vehicle before OVA challenge; n = 8 in each group. ∗, p < 0.05 compared with the vehicle-treated group. B, Airway responsiveness in mice treated with the IL-13 inhibitor or human IgG; n = 8 in each group. ∗, p < 0.05 compared with the IgG-treated group.

Discussion

In the present study, we investigated the in vivo interactions between IgE, mast cells, and CD8+ T cells, monitoring development of airway reactivity in mice passively sensitized with allergen-specific IgE and exposed to limited allergen challenge via the airways. The cumulative data suggest that allergen-specific CD8+ TEFF induce increased airway reactivity via a mechanism dependent on BLT1 and IL-13. In this study, we also showed that development of altered airway reactivity was dependent on mast cells and expression of FcεRI. Mast cell-deficient mice, similar to FcεRI−/− mice failed to increase BAL LTB4 levels following passive sensitization and challenge. Specifically, BLT1+/+ TEFF but not BLT1−/− TEFF mediate increased airway reactivity when transferred into CD8−/− recipient mice who showed increased levels of BAL LTB4 following passive sensitization and challenge. In addition, TEFF produced significantly more IL-13 compared with TCM following TCR cross-linking. Confirming the requirement for BLT1, LTB4 receptor blockade prevented the development of altered airway reactivity in BLT1+/+ reconstituted mice as well as in WT mice following passive sensitization and allergen challenge.

The role of mast cells in the development of increased airway reactivity is seemingly controversial. When studying mechanisms of allergic airway disease in animal models, mast cell-deficient mice have variable decreases in eosinophil numbers (31, 32) following allergen challenge, but systemically sensitized and challenged mast cell-deficient mice are capable of developing a Th2 response, airway inflammation, and AHR similar to WT control mice (33, 34, 35, 36). In most of these studies, allergen sensitization was achieved by systemically injecting the allergen in combination with an adjuvant, while other studies, using less potent sensitization protocols, demonstrated a more obvious role for mast cells in the development of AHR (37, 38). We have previously characterized a model of exclusive airway allergen exposure (over 10 consecutive days) in the absence of adjuvant and showed that the development of increased airway reactivity (measured as a response to EFS) is dependent on CD8+ T cells, mast cells, expression of FcεRI, and IL-13 production (6, 19). Mast cell activation led to increased levels of IL-13 in the lung. The IL-13 was not mast cell derived but was mast cell dependent and CD8 T cells appeared to be the primary source and were required for increased airway responsiveness to EFS. Other potential sources of IL-13 include CD4+ T cells, smooth muscle cells (39), basophils, and NK cells (40, 41). Recent reports have identified activated CD8+ lung T cells and TEFF as an important source of IL-13 (7, 30). The findings that CD8+ T cells are also necessary in this model for the induction of increased airway responsiveness is similar to the models following systemic sensitization (7).

In the present model of allergen-specific IgE passive sensitization and limited airway allergen exposure, transfer of allergen-specific TEFF to passively sensitized CD8−/− mice before limited allergen exposure induced an increased response to EFS. This only occurred when the recipient received allergen-specific IgE. Transfer of TEFF alone, in the absence of IgE with or without allergen exposure, failed to induce this increased responsiveness to EFS, suggesting that mast cell activation was indeed required for the induction of altered airway function. In contrast, transfer of allergen-specific TCM failed to induce increased airway responsiveness even when the recipient had been passively sensitized and allergen-challenged. There are several potential explanations for this disparity. One possibly relates to differences in capacity for production of IL-13, a cytokine central to the development of increased airway reactivity (3, 4, 42), especially in this model. Following stimulation through TCR, both do produce and secrete IL-13, however, the amounts of IL-13 secreted by TEFF were significantly higher than TCM. A second possibility could result from differences in the accumulation of TEFF and TCM in the lung. We showed that the numbers of TEFF in the lung (and BAL) were significantly higher than TCM following passive sensitization and challenge. This combination of greater numbers of TEFF in the lung and greater capacity for IL-13 production likely was the reason for the effectiveness of TEFF in the adoptive transfer experiments. The critical role of IL-13 was substantiated by the failure of IL-13−/− mice to develop AHR and importantly the effects of TEFF cell transfer could be abolished by treating recipients, including WT mice, with sIL-13Rα2, a potent inhibitor of IL-13 and AHR development (19, 24).

In trying to link the requirements for IgE, mast cells, CD8+ TEFF, and IL-13, we took advantage of the recent descriptions of LTB4 as a potent chemoattractant for TEFF cells (16, 17). Potential sources of LTB4 production in the lung are mast cells (43, 44, 45) as well as alveolar macrophages (46, 47), which can release this lipid mediator following cross-linking of FcRs. BLT1, the high-affinity receptor for LTB4, is induced substantially in CD8+ TEFF, with less expression on CD8+ TCM (16, 17). It has also been reported that LTB4 does elicit BLT1-dependent chemotaxis of TEFF, but not naive T cells or TCM (16, 17), suggesting the LTB4-BLT1 interaction as a potent nonchemokine pathway for TEFF accumulation. However, Tager et al. (48) reported the absence of differences in the migration of BLT1+/+ and BLT1−/− TEFF into the airways following adoptive transfer and airway challenge of naive (nonsensitized) recipients. In the present study, we showed that migration of TEFF into the airways following adoptive transfer and airway challenge of passively sensitized mice was significantly increased compared with adoptive transfer and challenge of naive mice, indicating that increased LTB4 production in the lung following sensitization and challenge is important for the “selective” migration of TEFF into the airways. Migration of BLT1−/− TEFF into the airways was significantly impaired compared with BLT1+/+ TEFF following passive sensitization and challenge and transfer of BLT1−/− T cells did not restore AHR. Pharmacological blockade of the LTB4 receptor confirmed these results as treatment with the LTB4 receptor antagonist prevented the induction of increased airway reactivity following TEFF transfer into passively sensitized and OVA-exposed CD8−/− mice as well as in WT mice. Other antagonists of the LTB4 receptor have also been reported to suppress AHR in rodent models (49, 50), consistent with the present data.

Coupled with the data on BAL LTB4 levels, these data support the notion that LTB4 production by mast cells following sensitization and challenge leads to migration of CD8+ TEFF cells into the airways through BLT1, and because of their capacity to produce IL-13, CD8+ TEFF mediate increased airway reactivity. In human asthma, BAL LTB4 levels have also correlated with disease severity (51) and the number of bronchial CD8 T cells has correlated with decline in lung function (52). In preliminary studies of BAL from asthmatics but not nonasthmatics, CD8+/BLT1+/IL-13+ T cells have now been identified (53).

In summary, we have demonstrated that CD8+ TEFF are capable of contributing to increased airway reactivity following mast cell activation in the lung, likely through their ability to produce IL-13 following their migration to the lung, which is, in turn, dependent on the IgE-FcεRI-mast cell-LTB4-BLT1 pathway. This suggests an important role for mast cells and activated CD8+ TEFF interactions in the development of AHR and the potential for targeting individual components of this pathway.

Acknowledgments

We thank D. Nabighian (National Jewish Medical and Research Center for her assistance.

Disclosures

The authors have no financial conflict of interest.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • ↵1 This work was supported by National Institutes of Health Grants HL-36577, HL HL-61005, HL-42246, and Environmental Protection Agency Grant R825702 (to E.W.G.). C.T. was supported by the Deutsche Forschungsgemeinschaft (Ta 275/2-1).

  • ↵2 C.T. and N.M. contributed equally to this work.

  • ↵3 Address correspondence and reprint requests to Dr. Erwin W. Gelfand, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206. E-mail address: gelfande{at}njc.org

  • ↵4 Abbreviations used in this paper: AHR, airway hyperresponsiveness; TCM, central memory CD8+ T cell; TEFF, effector CD8+ T cell; LTB4, leukotriene B4; WT, wild type; MNC, mononuclear cell; EFS, electric field stimulation; ES50, 50% of the maximal contraction; BAL, bronchoalveolar lavage.

  • Received August 22, 2005.
  • Accepted December 21, 2005.

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