Leukotriene B₄ Receptor 1 Expression on Dendritic Cells Is Required for the Development of Th2 Responses and Allergen-Induced Airway Hyperresponsiveness¹²

Asthma is characterized by persistent airway inflammation and airway hyperresponsiveness (AHR).⁵ Allergen-specific memory T cells that secret Th2-type cytokines such as IL-4, IL-5, and IL-13 are thought to play a central role in the development of the response (1, 2). A critical step in the induction of T cell activation is the uptake, processing, and presentation of Ag by APCs. Among the different types of APCs, dendritic cells (DC) play a key role for the initiation of immune responses in the airways. DCs recognize and take up Ag in the peripheral tissues, and process them. After Ag uptake and processing, DCs mature and migrate to secondary lymphoid organs via the activation of chemotactic receptors (3, 4). DCs then present processed peptides to the surface bound MHC molecules, which are recognized by T cells (5, 6), initiating T cell priming. It has been reported that extracellular stimuli such as chemokines and some lipid mediators such as prostaglandins and cysteinyl leukotrienes, stimulate DC chemotaxis (5, 6, 7). In animal models of allergic airway responses, DCs play crucial roles in both the sensitization and challenge phases (8, 9).

Leukotriene B₄ (LTB₄) is a proinflammatory lipid mediator which is derived from membrane phospholipids (10, 11, 12). LTB₄ activates leukocytes through a high-affinity G protein-coupled cell surface receptor, LTB₄ receptor 1 (BLT1), and leads to granulocyte and macrophage accumulation at sites of inflammation (13, 14, 15, 16, 17). We and others have recently shown that the LTB₄-BLT1 pathway may be important in effector T cell movement to sites of acute inflammation and Th2 cytokine production from T cells, and is critical to the development of AHR and inflammation (18, 19, 20, 21, 22, 23, 24). However, the effect of LTB₄ on allergic airway responses mediated through DCs in the airways has not been elucidated.

In this study, we investigated the role of BLT1 expression on DCs in the development of allergen-induced AHR and airway inflammation using mice that do not express this receptor. We found that murine lung DCs express BLT1 and that in an adoptive transfer model of allergen-induced airway responses, BLT1 expression on DCs was required for migration of DCs to regional lymph nodes as well as for the development of AHR and airway inflammation.

Female mice with a targeted disruption of the LTB₄ receptor 1 (BLT1^−/− mice) were backcrossed for seven generations onto a BALB/c genetic background (25). Leukotriene A₄ hydrolase-deficient (LTA₄H^−/−) mice and LTA₄H^+/+ mice (129/SvEv mice) were obtained from Dr. B. H. Koller (University of North Carolina, Chapel Hill, NC; Ref. 26). All mice were used at 8–12 wk of age. The mice were housed under specific pathogen-free conditions and maintained on an OVA-free diet in the Biological Resources Center 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.

DCs were generated from bone marrow cells of naive BLT1^+/+ or BLT1^−/− mice according to the procedure previously described (27). In brief, bone marrow cells obtained from femurs and tibias of mice were placed in culture medium with recombinant mouse GM-CSF (10 ng/ml) and recombinant mouse IL-4 (10 ng/ml; R&D Systems). Nonadherent cells were collected by aspirating the medium and transferred into fresh flasks. On day 8, cells were pulsed with OVA (grade V; Sigma-Aldrich; 200 μg/ml) for 24 h and washed three times with PBS. More than 90% of the cells were determined to be myeloid DCs (CD11c⁺, CD11b⁺, Gr-1⁻). BLT1^+/+ BMDCs or BLT1^−/− BMDCs (2 × 10⁵ cells) were incubated in 96-well culture plates with or without OVA (100 μg/ml). After 24 h, culture supernatants were harvested.

Under anesthesia, 1 × 10⁶ OVA-pulsed BMDCs in 40 μl of PBS were instilled into naive BLT1^+/+ mice through the trachea, under fiberoptic illumination (27). Control groups of mice received OVA-non-pulsed BMDCs. Ten days after BMDC transfer, mice were exposed to aerosolized OVA (1% in saline) for 20 min/day for three consecutive days; 48 h after the last challenge, AHR was assessed, and bronchoalveolar lavage (BAL) fluid and lung tissues were obtained.

To study the distribution of injected BMDCs, cells were labeled with the green fluorescent dye CFSE (Molecular Probes), as described previously (28). A total of 1 × 10⁶ BMDCs were instilled intratracheally into naive mice and traced 24, 48, and 120 h later in the BAL fluid, lung tissue, and peribronchial lymph nodes (PBLN). Samples were then analyzed by flow cytometry (FACSCalibur; BD Biosciences). In these experiments, mice were not exposed to aerosolized OVA after transfer of BMDCs.

DCs were also generated from bone marrow cells of naive LTA₄H^+/+ mice, and CFSE-labeled OVA-pulsed BMDCs were instilled into naive LTA₄H^+/+ or LTA₄H^−/− mice through the trachea. Distribution of the transferred BMDCs was evaluated in the same way.

Airway function was assessed as previously described by measuring changes in lung resistance (RL) and dynamic compliance (Cdyn) in response to increasing doses of inhaled methacholine (29). Data are expressed as percent change from baseline RL and Cdyn values obtained after inhalation of saline. There were no significant differences in baseline values among the different groups.

Immediately after assessment of AHR, 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 hematological procedures.

After BAL fluid was obtained, the lungs were fixed in 10% formalin and processed into paraffin blocks. Tissue sections 5 μm thick were stained with H&E and periodic acid-Schiff (PAS) for identification of mucus-containing cells. The number of mucus-containing cells per millimeter of basement membrane was determined as previously described (30).

Eosinophils were identified by immunohistochemistry using rabbit anti-mouse eosinophilic major basic protein Ab (kindly provided by Dr. J. J. Lee, Mayo Clinic, Scottsdale, AZ) as previously described (30). Numbers of peribronchial eosinophils in the tissues were evaluated as previously described (30).

PBLN were removed, and mononuclear cells (MNC) were purified by Ficoll-Hypaque gradient centrifugation (Organon Teknika) as previously described (31, 32). To evaluate cytokine production from T cells, T cells from PBLN MNC were isolated with a Mouse T Cell Recovery Column Kit (Cedarlane; purity >95%). T cells (2 × 10⁵) were cultured for 24 h in 96-well round-bottom plates in the presence or absence of OVA (100 μg/ml) with irradiated spleen cells (2 × 10⁵) as APCs.

Spleens of naive BLT1^+/+ mice were removed, MNC were first isolated by Ficoll-Hypaque gradient centrifugation, and then T cells were isolated with the Mouse T Cell Recovery Column Kit as described in PBLN cell preparations (purity >95%). Bone marrow-derived BLT1^+/+ or BLT1^−/− DCs were generated and pulsed with OVA as described in PBLN cell preparations, and isolated T cells (1 × 10⁶/ml) were cultured with OVA-pulsed BLT1^+/+ or BLT1^−/− BMDCs (0.5 × 10⁶/ml) for 4 days in 24-well flat-bottom tissue culture plates. Then, T cells were isolated with the Mouse T Cell Recovery Column Kit again, and 2 × 10⁵ cells were cultured for 24 h in 96-well round-bottom plates in the presence or absence of OVA (100 μg/ml) with irradiated spleen cells (2 × 10⁵) as APCs.

Spleens of OVA-sensitized BLT1^+/+ mice were removed, and MNC were first isolated by Ficoll-Hypaque gradient centrifugation, and then T cells were isolated with the Mouse T Cell Recovery Column Kit (purity >95%). Bone marrow-derived BLT1^+/+ or BLT1^−/− DCs were generated and pulsed with OVA as described in In vitro sensitization of naive T cells with OVA-pulsed BLT1^+/+ or BLT1^−/− DCs, and isolated (spleen) T cells (2 × 10⁵) were cultured with OVA-pulsed BLT1^+/+ or BLT1^−/− BMDCs (2 × 10⁴) for 72 h in 96-well round-bottom plates.

Cytokine levels in the BAL fluid and cell culture supernatants were measured by ELISA as previously described (28). IFN-γ, IL-4, IL-5, IL-10, IL-12 (BD Pharmingen), and IL-13 (R&D Systems) ELISAs were performed according to the manufacturer’s directions. The limits of detection were 4 pg/ml for IL-4, IL-5, IL-10, IL-12, and IL-13 and 10 pg/ml for IFN-γ.

Mouse anti-mouse BLT1 mAbs were generated by immunizing the BLT1^−/− mice (BALB/c background) with 300.19 cells expressing murine BLT1-red fluorescence protein as described elsewhere (S. Mathis et al., manuscript in preparation). Hybridoma culture supernatants of clone 3D7 were purified and fractionated over a Mono Q HR column (GE Healthcare). The peak fractions were biotinylated using the EZ-link sulfo-NHS-LC-biotin kit (Pierce) and used in flow cytometry.

To assess the migration of lung resident DCs from lung to PBLN, mice were anesthetized, and 100 μg of FITC-OVA in 50 μl of PBS were instilled intratracheally into naive BLT1^+/+ or BLT1^−/− mice. FITC⁺ DCs were traced 24 h later in the PBLN as previously described (33). Samples were analyzed by flow cytometry (FACSCalibur).

To evaluate BLT1 expression on BMDCs and lung DCs, mice were sensitized on days 1 and 14 by i.p. injection of 20 μg of OVA premixed with 2 mg of Al(OH)₃ (Pierce) in 100 μl of PBS. After sensitization, animals were exposed to aerosolized OVA (1% in saline) for 20 min/day on days 28, 29, and 30. Forty-eight hours after the last OVA challenge (day 32), MNCs from PBLNs and lung tissues were obtained. As a control, animals were injected with 100 μl of PBS followed by exposure on days 28–30 to aerosolized PBS. Biotinylated mouse anti-mouse BLT1 mAb and allophycocyanin-streptavidin (BD Pharmingen) were used to detect surface expression of murine BLT1. PE-conjugated anti-mouse CCR7 (eBioscience) was used to detect surface expression of murine CCR7. PE-conjugated or allophycocyanin-conjugated anti-mouse CD11c (BD Pharmingen) was used to identify DCs. PE-conjugated anti-mouse Gr-1 and allophycocyanin-conjugated CD11c were used to identify plasmacytoid DCs. The stained cells were washed, resuspended in PBS, and then analyzed by flow cytometry (FACSCalibur) as previously described (32). The numbers of CD11c⁺ and CFSE⁺ cells in BAL, PBLN, and lungs were derived by multiplying the percentage of stained cells by the total number of cells isolated.

All results were expressed as the means ± SEM. ANOVA was used to determine the levels of difference between all groups. Pairs of groups of samples distributed parametrically were compared by an unpaired two-tailed Student t test, and those samples distributed nonparametrically were compared by the Mann-Whitney U test. The data were pooled from three independent experiments with four mice per group in each experiment (n = 12). Significance was assumed at p values of <0.05.

We first assessed airway responsiveness to inhaled methacholine in mice that received BMDCs intratracheally. Intratracheal injection of OVA-pulsed BLT1^+/+ BMDCs followed by OVA challenge led to the development of increased AHR in recipient mice, illustrated by significant increases in RL and decreases in Cdyn, as compared with mice that received nonpulsed BMDCs (Fig. 1). In contrast, mice that received OVA-pulsed BLT1^−/− BMDCs developed lower increases in RL and decreases in Cdyn compared with the mice that received OVA-pulsed BLT1^+/+ BMDCs.

The numbers and types of inflammatory cells in the airways of the mice that received BLT1^−/− and BLT1^+/+ BMDCs were examined in BAL fluid (Fig. 2 A). After intratracheal transfer of OVA-pulsed BLT1^+/+ BMDCs and OVA challenge, the numbers of eosinophils in BAL fluid increased compared with those of the mice that received nonpulsed BMDCs. Eosinophil numbers in the BAL of mice that received OVA-pulsed BLT1^−/− BMDCs were significantly lower than in the mice that received OVA-pulsed BLT1^+/+ BMDCs.

Lung histology showed an intense infiltration of inflammatory cells in the perivascular tissue area and, to a slightly lesser extent, in the peribronchial spaces in the lungs of mice that received OVA-pulsed BLT1^+/+ BMDCs. In the mice that received OVA-pulsed BLT1^−/− BMDCs, fewer eosinophils were detected in these sites than in wild-type (WT) mice after challenge with OVA. Immunostaining with anti-major basic protein revealed significantly higher numbers of eosinophils in the peribronchial tissue of mice that received OVA-pulsed BLT1^+/+ BMDCs (mean ± SEM 112.2 ± 21.1 cells/mm²) when compared with mice that received OVA-pulsed BLT1^−/− BMDCs (65.9 ± 10.7 cells/mm²; p < 0.05) after challenge.

The degree of goblet cell metaplasia and mucus hyperproduction were assessed by PAS staining and quantification of PAS-stained cells. Transfer of OVA-pulsed BLT1^+/+ BMDCs into naive mice significantly increased the numbers of PAS⁺ cells when challenged with allergen (Fig. 2, B and C). However, mice that received OVA-pulsed BLT1^−/− BMDCs showed significantly fewer PAS⁺ goblet cells in the airways.

Concentrations of IL-4, IL-5, IL-13, and IFN-γ in the BAL fluid were measured by ELISA. Mice that received OVA-pulsed BLT1^+/+ BMDCs showed significantly increased levels of IL-4, IL-5, and IL-13 compared with the mice which received nonpulsed BMDCs (Fig. 2 D). The levels of IL-5 and IL-13 in the BAL fluid of the mice that received OVA-pulsed BLT1^−/−BMDCs were significantly lower than in recipients of OVA-pulsed BLT1^+/+ BMDCs.

We evaluated cytokine production in culture supernatants of BLT1^+/+ or BLT1^−/− BMDCs. The levels of IL-12 from OVA-pulsed BLT1^−/− BMDCs were similar to the levels from OVA-pulsed BLT1^+/+ BMDCs following restimulation with OVA (Fig. 2 E). The levels of IL-10 in the cultures of OVA-pulsed BLT1^−/− BMDCs were also similar to the levels in OVA-pulsed BLT1^+/+ BMDCs after restimulation with OVA.

We next determined if BLT1 participates in DC trafficking to PBLN. We first assessed BLT1 expression on established BMDCs and DCs from PBLN. As shown in Fig. 3,A, BMDCs expressed BLT1. To determine whether DCs in the PBLN expressed BLT1, MNC were isolated from the PBLN of OVA-sensitized and -challenged mice and then incubated with both anti-BLT1 and anti-CD11c. Gating on the CD11c⁺ population revealed that >80% of the cells stained positively for BLT1. At 24, 48, and 120 h after intratracheal instillation of OVA-pulsed CFSE-labeled BMDCs, MNC from the PBLN (BAL, and lung) were isolated, and transferred BMDCs were detected as CFSE⁺CD11c⁺ cells. Fig. 3 B illustrates the kinetics of appearance of BLTI^+/+ and BLT1^−/− CFSE⁺CD11c⁺ cells in the PBLN. The numbers of OVA-pulsed CFSE⁺BLT1^−/− BMDCs recovered from PBLN were significantly lower than from OVA-pulsed CFSE⁺BLT1^+/+ BMDCs at 24 and 48 h after instillation.

To determine whether the differences observed between the two strains of mice in their levels of Th2 cytokines were due to differences in Ag-specific T cell responsiveness, T cells obtained from PBLN were isolated and then restimulated in culture with OVA. The proportion of the cells that expressed CD3 exceeded 95%. IL-4, IL-5, and IL-13 production after culture with OVA from mice that received OVA-pulsed BLT1^−/− BMDCs was significantly lower than in the mice that received BLT1^+/+ BMDCs (Fig. 4).

Recipients of OVA-pulsed BLT1^−/− BMDCs developed lower AHR, airway inflammation, sensitization of T cells as well as the accumulation of transferred BMDCs in PBLN in vivo than did recipients of OVA-pulsed BLT1^+/+ BMDCs. To further investigate the mechanism underlying the impaired ability of BLT1^−/− DCs to prime T cells, we evaluated the functional activity of BLT1^+/+ and BLT1^−/− BMDCs in vitro. BLT1^−/− BMDCs showed normal maturation as judged by surface expression of CD80, CD86, MHC class II, CCR7, and CD40L (data not shown), consistent with a previous study (33). As shown in Fig. 5 A, when cultured with OVA-pulsed BLT1^−/− BMDCs, naive spleen T cells were able to produce comparable amounts of Th2 cytokines as cultures with OVA-pulsed BLT1^+/+ BMDCs. Naive T cells cultured with nonpulsed BLT1^+/+ or BLT1^−/− BMDCs did not increase the levels of Th2 cytokines (data not shown). These data suggested that BLT1^−/− DCs were not impaired in their ability to act as APCs in vitro.

DCs have been shown to be crucial not only in the sensitization phase but also during the effector phase (9). To determine whether BLT1 expression on DCs is essential for Ag presentation to activated T cells (as may occur in the allergen challenge phase), T cells from OVA-sensitized BLT1^+/+ mice were isolated and then cultured with OVA-pulsed BLT1^+/+ or BLT1^−/− BMDCs. As shown in Fig. 5 B, there were no differences in Th2 cytokine production from T cells obtained from OVA-sensitized mice when cultured with BLT1^+/+ BMDCs or BLT1^−/− BMDCs. Therefore, by all measures, BLT1^−/− DCs appeared to have normal Ag-presenting function, at least in vitro.

To determine whether the migration of DCs into PBLN was truly dependent on the LTB₄-BLT1 axis, we compared the accumulation of DCs in the PBLN of LTA₄H^+/+ and in LTA₄H^−/− mice, which lack LTB₄ production in vivo. We generated DCs from the bone marrow of LTA₄H^+/+ mice and then transferred OVA-pulsed CFSE-labeled BMDCs into LTA₄H^+/+ mice or LTA₄H^−/− mice intratracheally. The numbers of OVA-pulsed CFSE⁺ BMDCs recovered 24 and 48 h later from PBLN of LTA₄H^−/− mice were significantly lower than in LTA₄H^+/+ mice (Fig. 6). The numbers of CFSE⁺ BMDCs recovered in the BAL and lungs were not different in the two groups (data not shown).

The data illustrate that BMDCs express BLT1 and that the LTB₄-BLT1 pathway can regulate the migration of these cells to the PBLN. We next assessed the expression of BLT1 on lung DCs. As shown in Fig. 7, A and B, lung DCs in naive BLT1^+/+ mice express BLT1, and following systemic sensitization and challenge with OVA, the percentages of BLT1⁺ lung DCs significantly increased. After sensitization and challenge with OVA, the numbers of DCs in the lung of BLT1^−/− also increased, but to a significantly lesser extent than in BLT1^+/+ mice (Fig. 7 C).

CCR7 has been shown to play an important role in migration of DCs into regional lymph nodes. We determined whether BLT1 affected CCR7 expression on lung DCs. As shown in Fig. 7 D, percentages of CCR7⁺ DCs in the lung were significantly increased in BLT1^+/+ mice after sensitization and challenge with OVA, compared with naive BLT1^+/+ mice. The percentages of CCR7⁺ DCs in the lungs were significantly lower in BLT1^−/− mice than in BLT1^+/+ mice after sensitization and challenge.

The preceding data showed that BMDCs require the LTB₄-BLT1 pathway for migration to PBLN. We similarly assessed the migration of resident lung DCs to the PBLN to delineate the role of BLT1 in this process. To follow the migration of resident lung DCs from the lung to the PBLN, we gave mice 100 μg of FITC-OVA by intratracheal instillation. The phenotype and number of FITC-OVA⁺ cells in the PBLN were determined by flow cytometry 24 h after instillation. The numbers of FITC⁺ DCs in the PBLN of BLT1^−/− mice were significantly lower than the PBLN of BLT1^+/+ mice (Fig. 8,A). These data suggest that not only BMDCs but also lung DCs require BLT1 for migration to the PBLN. We also assessed the numbers of FITC⁺Gr-1⁺CD11c⁺ cells in the PBLN 24 h after instillation to determine whether migration of plasmacytoid DCs into PBLN was affected by BLT1 expression. Although lower in the BLT1^−/− mice, the numbers of FITC⁺Gr-1⁺CD11c⁺ cells in the PBLN were not significantly different in the two groups (Fig. 8 B).

Previous studies have shown that LTB₄ plays a central role in the early recruitment of leukocytes and Ag-specific effector memory CD8⁺ T cells and Th2 cytokine production in allergen-induced airway responses (19, 20, 21, 22, 23, 24). However, the role for this receptor on DCs in the development of AHR and inflammation has not been defined. Evidence for expression of a functional BLT1 receptor on DCs was provided by Del Prete et al. (34), who showed that murine DCs mobilize calcium and chemotaxis in response to LTB₄ and that BLT1 expression on DC regulates delayed-type hypersensitivity in the skin. Here, we confirm that DCs in the lungs, regional lymph nodes, and airways express BLT1 and that expression of BLT1 on DCs can play an important role in the development of allergen-induced AHR and inflammation. Moreover, after sensitization and challenge, the numbers of lung DCs expressing BLT1 increased significantly over the numbers seen in naive BLT1^+/+ mice or the numbers from sensitized and challenged BLT1^−/− mice. Using a BMDC-dependent model of allergic airway disease, AHR and airway inflammation in mice that received BLT1^−/− DCs were significantly reduced compared with the mice that received BLT1^+/+ DCs. After transfer, migration of BLT1^−/− DCs to the regional lymph nodes was significantly impaired compared with transfer of BLT1^+/+ DCs. In vivo as well as in vitro Th2 cytokine production from PBLN T cells was significantly reduced in the recipients of BLT1^−/− DCs compared with mice that received BLT1^+/+ DCs. These data suggest that BLT1 expression on DCs plays an important role in the development of AHR and inflammation, at least in part facilitated by their ability to migrate to the regional lymph nodes.

The importance of the migration of lung DCs into regional lymph nodes for the development of allergen-induced airway responses has been suggested (35, 36). They found that during the sensitization phase, DCs in the lungs migrate into the regional lymph nodes, where initial priming of T cells occurs. After airway challenge, sensitized T cells produce Th2 cytokines and induce AHR and eosinophilic inflammation in the airways. Therefore, any interference with the migratory capacity of DCs to the regional lymph nodes could result in impairment of the initial sensitization of T cells, and consequently lower eosinophilic inflammation and AHR. In the present study, the absence of BLT1 on DCs resulted in impaired migration to the regional lymph nodes after intratracheal instillation. To confirm that the migration of DCs to the lymph nodes was dependent on the LTB₄-BLT1 pathway, we evaluated the ability of DCs from WT mice to accumulate in the regional lymph nodes of LTA₄H^−/− mice, i.e., in an environment where LTB₄ production was completely lacking. DC accumulation in the regional lymph nodes was markedly decreased in the deficient mice, further supporting the notion that recruitment of DCs to the regional lymph nodes in the airways is dependent on the LTB₄-BLT1 pathway. To complement and extend these findings, we tracked lung DC migration after intratracheal instillation of FITC-OVA. In these studies, we confirmed that BLT1 expression on lung DCs plays an important role in their migration to the regional lymph nodes. The impairment associated with the absence of BLT1 expression appeared restricted to DC migratory capacity but not on other aspects that may have been associated with impaired DC function. BLT1^−/− BMDCs showed normal maturation as judged from the expression of surface markers. Further, in vitro, BLT1^−/− BMDCs were fully capable of activating naive spleen T cells to produce Th2-type cytokines, similar to BLT1^+/+ BMDCs.

In the process of DC migration to lymph nodes, chemokine signals acting on the CCR7 are important. In mice homozygous for the paucity of lymph node T cell (plt) mutation which lack the CCR7 agonists CCL19 and CCL21, migration was similarly impaired (37). The migration of Ag-pulsed DCs into peripheral tissues has been shown to be dependent on the expression of CCR7 by DCs and the production of CCR7 ligands, CCL19, by stromal cells and mature DCs in the lymph node, and CCL21 by afferent lymphatic cells. Del Prete et al. reported that LTB₄ up-regulates CCR7 expression and function and that this effect is primarily through the enhancement of chemotaxis with only a minor effect on chemokinesis (34). They showed that chemotaxis of BLT1^−/− BMDCs in response to CCL19 and CCL21 was significantly impaired compared with BLT1^+/+ BMDCs, that LTB₄-induced migration of immature DCs to CCL19 and CCL21 was associated with an increase in CCR7 membrane expression, and that LTB₄ up-regulates the migration of mature DCs to CCR7 ligands. In the present study, expression of CCR7 on lung DCs was higher in BLT1^+/+ mice compared with BLT1^−/− mice following sensitization and challenge conditions where LTB₄ levels were also increased (12). Therefore, it appears that LTB₄ can up-regulate CCR7 expression, which in turn, leads to the migration of DCs to regional lymph nodes. Migration of DCs into regional lymph nodes has also been reported to be controlled by other lipid mediators, such as prostaglandins and cysteinylleukotrienes. PGD2 acts on the DP1 receptor expressed by lung DCs and suppresses the migration of lung DCs (38). The leukotriene C₄ transporter, MRP1, regulates DC migration to lymph nodes by transporting leukotriene C₄, which in turn promotes chemotaxis to CCL19 and mobilization of DCs from the epidermis (39).

Von Rijt et al. (9) showed that depletion of CD11c⁺ cells during allergen challenge in sensitized mice significantly suppressed features of asthma in mice. These data suggest that during the allergen challenge phase, DCs also migrate from the sites of allergic inflammation to the draining lymph nodes to induce further expansion of recirculating central memory cells or to stimulate naive T cells to become Th2 cells, thus enhancing and sustaining the inflammatory response with new waves of effector T cells (40, 41, 42). In the present study, allergen-sensitized T cells restimulated with allergen-pulsed BLT1^−/− DCs were able to produce Th2 cytokines similar to those restimulated with BLT1^+/+ DCs, suggesting that Ag-presenting function during the restimulation phase was not impaired, at least in vitro. Thus, it is more likely that in recipients of BLT1^−/− DCs, impaired migration in the allergen challenge phase in vivo might have also contributed to the development of lower levels of AHR, inflammation, and Th2 cytokines compared with these mice which received BLT1^+/+ DCs.

In this study, we utilized a well-characterized DC-dependent model for development of allergen-induced airway responses and found that in the absence of BLT1 expression on DCs the full spectrum of lung allergic responses was decreased: eosinophilic inflammation; Th2 cytokine production; AHR; and goblet cell metaplasia. These findings are supported by our previous results demonstrating that BLT1^−/− mice showed significantly lower AHR and goblet cell metaplasia than did BLT1^+/+ mice, although they did not show significantly lower eosinophilic inflammation than did BLT1^−/− mice when systemically sensitized with OVA together with alum (21). Full reconstitution of all responses in these BLT1^−/− recipients was demonstrated after transfer of Ag-primed BLT1^+/+ T cells (20). Reconstitution with Ag-primed T cells or T cell subsets likely bypasses some of the requirements for BLT1 expression in the BLT1^−/− recipients. Further, in the present study, we did not use any adjuvant for sensitization and in fact bypassed the sensitization phase rendering the system totally dependent on transfer of Ag-pulsed DCs. It has been shown that adjuvant enhances expression of MHC class II and costimulation molecules such as CD80 and CD86 and up-regulates the Ag-presenting function of DCs (43, 44). Therefore, in the earlier study, systemic sensitization to allergen together with alum may have bypassed some aspects of DC function dependent on BLT1 expression, accounting for the differences in eosinophilic inflammation between BLT1^−/− and BLT1^+/+ mice and enabling reconstitution of sensitized BLT1^−/− recipients with primed CD8⁺ T cells.

In summary, we have identified the critical role for a functional LTB₄-BLT1 pathway in DCs for the full development of allergen-induced AHR and airway inflammation, and have shown that BLT1 expression on DCs was required for the full development of these responses. Our studies indicate that BLT1 controls migration of DCs into the regional lymph nodes, the initiation of T cell responses leading to Ag-specific Th2 responses, and the development of AHR and airway inflammation. Manipulating DC function through BLT1 may provide a novel approach for controlling allergen-induced AHR and eosinophilic airway disease and has the potential to improve the treatment of asthma.

We thank L. N. Cunningham and D. Nabighian (National Jewish Medical and Research Center) for assistance.

The authors have no financial conflict of interest.