Recent studies in both human and rodents have indicated that in addition to CD4+ T cells, CD8+ T cells play an important role in allergic inflammation. We previously demonstrated that allergen-sensitized and -challenged CD8-deficient (CD8−/−) mice develop significantly lower airway hyperresponsiveness (AHR), eosinophilic inflammation, and IL-13 levels in bronchoalveolar lavage fluid compared with wild-type mice, and that all these responses were restored by adoptive transfer of in vivo-primed CD8+ T cells or in vitro-generated effector CD8+ T cells (TEFF). Recently, leukotriene B4 and its high affinity receptor, BLT1, have been shown to mediate in vitro-generated TEFF recruitment into inflamed tissues. In this study we investigated whether BLT1 is essential for the development of CD8+ T cell-mediated allergic AHR and inflammation. Adoptive transfer of in vivo-primed BLT1+/+, but not BLT1−/−, CD8+ T cells into sensitized and challenged CD8−/− mice restored AHR, eosinophilic inflammation, and IL-13 levels. Moreover, when adoptively transferred into sensitized CD8−/− mice, in vitro-generated BLT1+/+, but not BLT1−/−, TEFF accumulated in the lung and mediated these altered airway responses to allergen challenge. These data are the first to show both a functional and an essential role for BLT1 in allergen-mediated CD8+ TEFF recruitment into the lung and development of AHR and airway inflammation.
Allergic asthma is a complex syndrome that has been characterized by airway obstruction, airway inflammation, and airway hyperresponsiveness (AHR)3 (1, 2). Ag-specific memory T cells, especially CD4+ T cells, have been considered pivotal in the development of AHR and eosinophilic inflammation (1, 2, 3, 4) through the production of cytokines, especially IL-13 (5, 6). However, there is now increasing evidence for the role of CD8+ T cells in these responses as well. Increased numbers of CD8+ T cells have been shown in the lungs of asthmatic patients (7) and in animal models of allergic asthma (8). In addition, CD8+ T cells in the lung are a source of IL-13 and may be required for the full development of allergen-induced AHR and inflammation (9). Moreover, in vitro-generated Ag-specific effector CD8+ T cells (TEFF) contribute to these responses through their migration into lung tissue and local production of IL-13 in sensitized and challenged mice (10).
Leukotriene B4 (LTB4) is an arachidonic acid-derived proinflammatory lipid, rapidly generated from innate immune cells (11, 12). LTB4, interacting through a specific G protein-coupled cell surface receptor, BLT1 (13, 14), leads to granulocyte and macrophage accumulation at sites of inflammation (15, 16). Because BLT1 is expressed by TEFF, the LTB4-BLT1 pathway may be essential for effector CD8+ T cell movement to sites of acute inflammation (17, 18). However, its role in the development of allergen-induced AHR and inflammation has not been defined.
In the present study we investigated the requirement for BLT1 expression on CD8+ T cells and Ag-specific TEFF in the development of allergen-induced AHR and airway inflammation. We show that BLT1-deficient (BLT1−/−) CD8+ T cells or BLT1−/− TEFF are not capable of restoring these responses in CD8−/− mice. The absence of BLT1 resulted in the reduced accumulation of CD8+ T cells in the lungs and decreased IL-13 production in sensitized and challenged CD8−/− mice. These data demonstrate that BLT1 is essential for allergen-mediated CD8+ T cell recruitment to the lung and induction of eosinophilic airway inflammation and AHR.
Materials and Methods
Homozygous CD8−/− mice, generated by targeting the CD8 α-chain gene in C57BL/6 mice (19), OT-1 mice (C57BL/6 strain) expressing a transgenic TCR that is specific for OVA257–264 (SIINFEKL) peptide (20), and wild-type (WT) C57BL/6 mice were purchased from The Jackson Laboratory. BLT1−/− mice, F1 hybrids of C57BL/6 and 129Sv/J genetic background (21), 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. These 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. Male and female mice, 6–12 wk of age, were used in these experiments. Controls were matched with the deficient mice with regard to age and gender in each experimental group. 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.
Sensitization and airway challenge
CD8−/− and WT mice were assigned to control (C) and treatment groups (S/C) based on the following treatments: 1) airway challenge with OVA nebulization alone (three times; C group), and 2) i.p. sensitization with OVA and OVA airway challenge (S/C). Mice were sensitized by i.p. injection of 20 μg of OVA (grade V; Sigma-Aldrich) emulsified in 2.25 mg of alum (AlumImuject; Pierce) on days 1 and 14. Mice were subsequently challenged via the airways by inhalation exposure to aerosols of OVA (1% in saline) for 20 min on days 28, 29, and 30. OVA aerosols were produced by an ultrasonic nebulizer (particle size, 1–5 μm; De Vilbiss). On day 32, airway function was measured as described below, followed by collection of samples for further analyses.
Purification of CD8+ T cells
Spleens of BLT1−/− mice or BLT1+/+ mice, which were sensitized twice (days 1 and 14) with OVA plus alum, were removed 14 days after the last sensitization (day 28), and purification of CD8+ T cells was performed by negative selection using MACS (Miltenyi Biotec) as previously described (9). To assess purification, cells were incubated with allophycocyanin-conjugated anti-CD3; FITC-conjugated anti-CD4, anti-CD8, or anti-B220 Abs; and PE-conjugated anti-CD11c, anti-mouse NK1.1, anti-γδ TCR, or anti-αβ TCR (BD Pharmingen), then analyzed by flow cytometry (FACSCalibur; BD Biosciences). The proportion of the transferred cells that stained for CD3+CD8+ from BLT1+/+ or BLT1−/− mice exceeded 93%. They were >99% αβ+CD8+ T cells, with <0.5% of cells being γδ+CD8+ T cells in preparations from both groups. Contamination by CD4+ cells, γδ+ cells, CDllc+ cells, or NK1.1+ cells in transferred cells was, in total, <0.5%.
Effector CD8+ T cell generation
Differentiation of TEFF cells in vitro was conducted as previously described (10). Lymph node and spleen cells collected from BLT1+/+ and BLT1−/− OT-1 TCR transgenic mice were cultured with 1 μM SIINFEKL peptide. After culture for 2 days, cells were washed and cultured with IL-2 (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+ effector memory cell phenotype, as shown by the CD122high/CD44high/CD62Llow cell surface phenotype. TEFF derived through this protocol showed phenotypic and functional characteristics of effector memory CD8+ T cells in vivo (22, 23).
Adoptive transfer recipient mice
Recipient CD8−/− mice were sensitized twice with OVA plus alum on days 1 and 14. OVA-primed CD8+ T cells (5 × 106) from BLT1+/+ or BLT1−/− mice were administered i.v. via the tail vein to OVA-sensitized CD8−/− mice 14 days after the last sensitization (day 28). In vitro-generated CD8+ TEFF cells (5 × 106) from BLT1+/+ or BLT1−/− mice were transferred in the same way. After transfer, the mice were exposed to three allergen challenges via the airways on days 28, 29, and 30. Assays were conducted on day 32.
Assessment of airway function
Airway function was assessed as previously described by measuring changes in lung resistance (RL) in response to increasing doses of inhaled methacholine (24). Data are expressed as the percentage of change from baseline RL values obtained after inhalation of saline.
Bronchoalveolar lavage (BAL)
Immediately after assessment of AHR, lungs were lavaged via the tracheal tube with HBSS. Total leukocyte numbers were counted by cell counter (Coulter). Cytospin slides were stained with Leukostat (Fisher Diagnostics) and differentiated by standard hematological procedures.
Measurement of cytokines
Cytokine levels in the BAL fluid and cell culture supernatants were measured by ELISA as previously described (25). IFN-γ, IL-4, IL-5 (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, and IL-13 and 10 pg/ml for IFN-γ.
Mononuclear cells (MNC) from spleen and peribronchial lymph node (PBLN) were purified by Ficoll-Hypaque gradient centrifugation (Organon Teknika) (9). Lung cells were isolated as previously described using collagenase digestion (26). Cells were resuspended in HBSS, and MNC were purified by Ficoll-Hypaque gradient centrifugation.
After purification, 1 × 106 cells were incubated with allophycocyanin-conjugated anti-CD3 and FITC-conjugated anti-CD8 Abs (BD Pharmingen), then analyzed by flow cytometry (FACSCalibur; BD Biosystems) as previously described (9). The number of CD8+ T cells per lung was derived by multiplying the percentage of stained cells by the total number of lung cells isolated. Intracytoplasmic cytokine staining for IFN-γ (BD Pharmingen) and IL-13 (R&D Systems) was performed as previously described (9). The number of cytokine-producing CD8+ T cells per lung was calculated from the percentage of cytokine-producing cells and the number of CD8+ T cells isolated from the lung.
Values for all measurements are expressed as the mean ± SEM. ANOVA was used to determine the levels of difference between all groups. Comparisons for all pairs were performed by unpaired two-tailed Student’s t test. Significance levels were set at p < 0.05.
In vivo-primed CD8+ T cells from BLT1+/+, but not BLT1−/−, mice fully restore AHR in CD8−/− mice
Fig. 1,A shows the changes in RL in response to increasing doses of inhaled methacholine for both WT and CD8−/− mice. OVA sensitization and airway challenge led to the development of increased AHR in WT mice, illustrated by significant increases in RL, as previously described in this model (23). OVA-sensitized and -challenged CD8−/− mice developed small increases in RL above nonsensitized and OVA-challenged control mice, but the levels were significantly lower than those in OVA-sensitized and -challenged WT mice (9). To address whether the expression of BLT1 on CD8+ T cells is involved in the regulation of allergen-induced AHR, inflammation and cytokine responses, CD8+ T cells from BLT1+/+ and BLT1−/− mice were injected into CD8−/− mice. Recipient CD8−/− mice were sensitized with OVA on days 1 and 14. On day 28, 2 h before beginning the OVA challenges, 5 × 106 CD8+ T cells from BLT1+/+ or BLT1−/− mice were transferred via i.v. injection into the tail vein. As shown in Fig. 1 A, reconstitution of CD8−/− mice with Ag-primed CD8+ T cells from BLT1+/+ mice fully restored the development of AHR to levels comparable to those seen in WT mice. In contrast, transfer of OVA-sensitized CD8+ T cells from BLT1−/− mice failed to restore AHR.
In vivo primed CD8+ T cells from BLT1+/+ mice, but not BLT1−/−, mediate allergen-induced eosinophilic airway inflammation
Previous studies indicated that CD8−/− mice failed to develop OVA-induced eosinophil accumulation in BAL, a response that was restored after adoptive transfer of Ag-primed CD8+ T cells (9). After sensitization and allergen challenge, the numbers of eosinophils in the CD8−/− mice were significantly lower than those in WT mice (Fig. 1 B). Transfer of BLT1+/+ CD8+ T cells fully restored the number of eosinophils in BAL fluid, whereas BLT1−/− CD8+ T cell transfer failed to do so.
Cytokine levels in BAL fluid
OVA sensitization and challenge did not result in significant differences between WT and CD8−/− mice in the levels of IL-4, IL-5, or IFN-γ in BAL fluid (9). However, after sensitization and challenge, the levels of IL-13 were significantly lower in BAL fluids recovered from CD8−/− mice compared with WT mice (Fig. 1 C). Reconstitution of CD8−/− mice with CD8+ T cells from BLT1+/+, but not BLT1−/−, restored the levels of IL-13 in BAL fluid to those seen in WT mice.
Migration of BLT1+/+ and BLT1−/− CD8+ T cells
We next determined whether transferred, in vivo-primed CD8+ T cells migrated to the lung 48 h after challenge of sensitized CD8−/− mice. Under this background, transferred cells were easily identified as CD3+CD8+ cells. Transferred BLT1+/+ CD8+ T cells amounted to 1.2 ± 0.2% (mean ± SEM) of the total T cells in lungs after OVA sensitization and challenge, whereas transferred BLT1−/− CD8+ T cells were found at a lower percentage (0.6 ± 0.1% of total lung T cells). Fig. 1 D summarizes the numbers of transferred CD8+ T cells in the lungs, BAL, PBLN, and spleens of recipient mice. Higher numbers of transferred BLT1+/+ CD8+ T cells were recovered from lung and PBLN compared with BLT1−/− CD8+ T cells; the numbers in BAL and spleen were not different between the two groups. Together, these data identify a role for BLT1 in the accumulation of CD8+ T cells in the lung after sensitization and challenge.
BLT1+/+, but not BLT1−/−, TEFF fully restores AHR in CD8−/− mice
We previously showed that transfer of CD8+ TEFF, but not central memory CD8+ T cells (TCM), fully restored AHR and cytokine responses in CD8−/− recipients (9). To address whether the expression of BLT1 on CD8+ TEFF is required for these airway and cytokine responses, CD8−/− mice received in vitro-generated BLT1-deficient or BLT1-sufficient CD8+ TEFF cells. Fig. 2,A shows that reconstitution of the CD8−/− mice with TEFF from BLT1+/+ mice fully restored AHR to levels comparable to those seen in WT mice. In contrast, transfer of BLT1−/− TEFF failed to restore AHR. Transfer of BLT1−/− TEFF also failed to restore the number of eosinophils and IL-13 levels in the BAL; transfer of BLT1+/+ TEFF fully restored these responses (Fig. 2, B and C).
Migration of transferred TEFF cells into lung
The accumulation of adoptively transferred TEFF in the lungs of CD8−/− mice was determined 48 h after the last challenge. Transferred BLT1+/+ TEFF cells (identified as CD3+CD8+) amounted to 11.0 ± 2.5% of the total lung MNC after OVA sensitization and challenge (Fig. 3,A), whereas transferred BLT1−/− TEFF cells were found at a much lower percentage (2.0 ± 0.3% of total lung MNC). A similar pattern was observed in BAL fluid (Fig. 3,A). More than 95% of the BLT1+/+ and BLT1−/− TEFF in lung and BAL displayed an effector memory phenotype: CD122high/CD44high/CD62low (22). Fig. 3 B summarizes the numbers of transferred TEFF detected in the lungs, PBLN, BAL, and spleen of recipient CD8−/− mice. Lower numbers of BLT1−/− TEFF were detected in BAL and lung, whereas higher numbers of transferred BLT1−/− TEFF cells were recovered from PBLN.
Intracellular cytokine detection in transferred TEFF
In addition to or as a consequence of the marked reduction in recruitment of BLT1−/− TEFF, we determined whether the failure to restore AHR and cytokine response by transferred BLT1−/− TEFF could also be due to decreased IL-13 production by these cells. MNCs were obtained from lung tissue of recipient CD8−/− mice after transfer of either BLT1+/+ TEFF or BLT1−/− TEFF 48 h after the last challenge. The cells were stimulated with PMA/ionomycin and incubated with Abs for intracellular staining of IL-13 as described in Materials and Methods. Fig. 4 A illustrates the percentage of cytokine-producing TEFF in recipient lungs. IL-13 was detected in 33.3 ± 2.8% of transferred BLT1+/+ TEFF cells, whereas among the BLT1−/− TEFF cells, only 13.8 ± 1.5% expressed IL-13. After in vitro culture and just before transfer, there were no significant differences in IL-13 production between BLT1+/+ and BLT1−/− TEFF (5.2 ± 1 vs 4.7 ± 0.9%).
In parallel, we monitored CD4+ T cell numbers and cytokine production. Numbers of CD4+ T cells in the lungs of BLT1−/− TEFF recipient mice were not different compared with those in BLT1+/+ TEFF recipient mice. Similarly, the numbers of IL-4- and IL-5-producing CD4 T cells in the lungs of BLT1−/− TEFF recipient mice were not different from those in BLT1+/+ TEFF recipient mice. These data suggest that CD4 recruitment or activation in the lung is not affected by the expression of BLT1.
These results demonstrate that in addition to the reduced recruitment, a smaller percentage of the transferred BLT1−/− TEFF cells was capable of IL-13 production after airway allergen challenge of sensitized recipients. Overall, the numbers of IL-13-producing BLT1−/− TEFF cells were significantly lower than the numbers of IL-13-producing BLT1+/+ TEFF cells (Fig. 4, B and C). By contrast, the percentage of IFN-γ-producing cells did not change. These data illustrate that in addition to its role in cell trafficking to the lung, the functional activation of TEFF and IL-13 production may be dependent in part on the expression of BLT1.
There is now increasing evidence that CD8+ T cells contribute to the development of allergic disease (7, 8, 9, 10), but their function in allergic airway disease, especially how they are recruited and activated in the lung, is not well defined. LTB4 has been shown to be a potent chemoattractant for effector CD8+ T cells, and LTB4 production by activated leukocytes, such as neutrophils, mast cells, and macrophages, may be important in the recruitment of TEFF cells to sites of inflammation (17, 18, 27, 28). BLT1, the high affinity receptor for LTB4, has been induced substantially on CD8+ TEFF, with less expression on CD8+ TCM (17). LTB4 triggers BLT1-dependent chemotaxis of TEFF, but not naive T cells or TCM (17, 27), suggesting that the LTB4-BLT1 interaction is a potent nonchemokine pathway for effector CD8+ T cell accumulation.
In the present study we investigated the role of this LTB4-BLT1 pathway in effector CD8+ T cell function in a mouse model of allergen-induced AHR and airway inflammation. We have reported that CD8−/− mice develop significantly lower AHR and eosinophilic inflammation compared with WT mice, and that these deficient responses were restored by transfer of in vivo-activated CD8+ T cells from Ag-primed donors or by transfer of in vitro-generated CD8+ TEFF, identifying a critical role for CD8+ T cells (9, 10). In the present study we prepared in vivo-primed CD8+ T cells and in vitro-generated, Ag-specific CD8+ TEFF from BLT1−/− mice to address the question of whether BLT1 expression on CD8+ T cells affects their migration and effector function and, consequently, the development of AHR and airway inflammation. After transfer of these cells into CD8−/− mice, a significant number of the transferred BLT1+/+ CD8+ T cells and BLT1+/+ TEFF cells were detected in the lung, and this was associated with the development of AHR, eosinophilia, and IL-13 production. Transfer of BLT1−/− CD8+ T cells or BLT1−/− TEFF cells was not effective in mediating any of these responses. This appeared to be due to the absence of two components of the response: 1) failure to accumulate in the lung, and 2) decreased functional activation in the lung, at least for IL-13 production. These two deficiencies probably explain why transfer of CD8+ T cells and TEFF cells from BLT1+/+ mice enhanced IL-13 levels in BAL fluid and restored AHR, whereas BLT1−/− cells did not. Furthermore, we demonstrated that although numbers of IL-13-producing BLT1+/+ TEFF in the lung after transfer were significantly higher than those of BLT1−/− TEFF, the numbers of IL-13-producing BLT1+/+ TEFF in vitro before transfer were not different from those of BLT1−/− TEFF. These data suggest that BLT1+/+ TEFF responded in the lung after sensitization and challenge and acquired effector function (IL-13 production), whereas BLT1−/− TEFF failed to do so. These data demonstrate for the first time, in an in vivo model, that migration/accumulation as well as the functional activation of TEFF in vivo are at least in part dependent on the LTB4-BLT1 pathway. Together, the data indicate a critical role for BLT1-effector CD8+ T cells in the full development of AHR and airway inflammation. In contrast, CD4+ T cell numbers and cytokine production were not affected by the absence of BLT1.
TEFF have been reported to produce IFN-γ in vitro (22, 29). However, after adoptive transfer, TEFF in the lung displayed a Tc2 phenotype (10). After in vitro stimulation with IL-4, cytotoxic CD8+ T cells can be polarized to Th2-type cytokine-producing cells (30, 31, 32). Similarly, virus-specific CD8+ T cells may convert into IL-5-producing cells in mice sensitized to OVA plus alum, followed by challenge with virus peptide (33). Thus, the phenotype of even predominant Th1-type cytokine-producing CD8+ T cells may be redirected toward Th2-type cytokine production, a plasticity previously emphasized in CD4+ T cells (34).
IL-13 plays a central role in the development of AHR (6, 7). We showed that lung CD8+ T cells are a source of IL-13, and reconstitution of CD8−/− mice with IL-13−/− CD8+ T cells failed to restore AHR, confirming that CD8+ T cells can contribute to the development of AHR and airway inflammation through IL-13 production (9). LTB4 is known to augment T cell cytokine secretion in vitro (35, 36, 37, 38, 39). In the present study we showed that IL-13 production from CD8+ T cells may require BLT1 expression on effector CD8+ T cells. Interestingly, airway responses induced by rIL-13 may also require an intact LTB4 pathway in vivo (40). Because CD8+ TEFF are a source of IL-13 after Ag challenge, this production of IL-13 may further activate LTB4 production in the lung and serve to amplify or enhance the accumulation and activation of Tc2-type effector CD8+ T cells.
Tager et al. (18) reported that BLT1 participates in T cell trafficking into the BAL, but not lung tissue, at an early phase after challenge of sensitized mice. In contrast, TEFF did not require this receptor for trafficking into the airway after adoptive transfer and airway challenge of naive (nonsensitized) recipients (18). In contrast, using sensitized, as opposed to naive, recipient mice, we show that migration of transferred BLT1−/− TEFF into lung as well as BAL was significantly impaired compared with that of BLT1+/+ TEFF. In this adoptive transfer model, the recipient mice were sensitized to OVA (plus alum) before OVA challenge. LTB4 production in the lungs of sensitized and challenged recipients should be significantly higher than that of challenged-only recipients (28), and these increased levels of LTB4 may play a pivotal role in enhancing the recruitment of transferred BLT1+/+ TEFF into the lung.
Different members of the chemokine family are known to be subset-selective chemoattractants for T cells (41). For example, it has been shown that CCL2 and CCL5 may be important in the recruitment of CD8+ T cells (42). TEFF were reported to migrate in response to CCL5 as well as LTB4 in vitro (27). In our transfer experiments we demonstrated that the migration of both BLT1−/− CD8+ T cells and BLT1−/− CD8+ TEFF into the lung was significantly impaired compared with that of BLT1+/+ cells; this impairment in the TEFF was more obvious than that in CD8+ T cells. In the latter case, only small numbers of Ag-specific CD8+ T cells are probably generated compared with TEFF. These results indicate that although the LTB4-BLT1 pathway regulates CD8+ T cell recruitment, it is essential for trafficking of TEFF to sites of airway inflammation in vivo. LTB4-dependent signals contribute at least one essential link to a chain of molecular events that may be required for efficient recruitment of TEFF.
Several cellular constituents of the innate immune system are capable of generating LTB4 (12, 13). Mast cells are a major source of lipid-derived mediators, and activation of mast cells through FcεRI stimulates rapid degranulation and release of LTB4 (43). LTB4 produced by mast cells induces chemotaxis of TEFF in vitro (27); therefore, after sensitization and challenge with Ag, production of LTB4 from mast cells is probably a major contributor to TEFF recruitment to inflamed tissues. LTB4 production from cells other than mast cells, such as neutrophils and macrophages (12, 13), may similarly be capable of recruiting TEFF to the lung in this model.
In summary, we have identified a critical role for BLT1 expression on effector CD8+ T cells in the development of allergen-induced AHR and airway inflammation. In vivo, BLT1 expression on effector CD8+ T cells may be required for their migration and effector function and the full development of airway eosinophilia and AHR. TheTEFF-BLT1 pathway may constitute a novel therapeutic target in bronchial asthma.
The authors have no financial conflict of interest.
We thank L. N. Cunningham and D. Nabighian (National Jewish Medical and Research Center) for their assistance.
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.
This work was supported by National Institutes of Health Grants HL36577, HL61005, and AI42246, and by Environmental Protection Agency Grant R835702.
Abbreviations used in this paper: AHR, airway hyperresponsiveness; BAL, bronchoalveolar lavage; BLT1, leukotriene B4 receptor 1; LTB4, leukotriene B4; MNC, mononuclear cell; PBLN, peribronchial lymph node; PI, phorbol myristate acetate/ionomycin; RL, lung resistance; TCM, central memory CD8+ T cell; TEFF, effector CD8+ T cell; WT, wild type.