HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Miyahara, N. Articles by Gelfand, E. W. Search for Related Content PubMed PubMed Citation Articles by Miyahara, N. Articles by Gelfand, E. W.

Leukotriene B4 Receptor-1 Is Essential for Allergen-Mediated Recruitment of CD8⁺ T Cells and Airway Hyperresponsiveness¹

Division of Cell Biology, Department of Pediatrics, National Jewish Medical and Research Center, Denver, CO 80206; and Center for Immunology and Inflammatory Diseases, Division of Rheumatology, Allergy and Immunology, Massachusetts General Hospital, Charlestown, MA 02129

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
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 (T_EFF). Recently, leukotriene B4 and its high affinity receptor, BLT1, have been shown to mediate in vitro-generated T_EFF 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^–/–, T_EFF 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⁺ T_EFF recruitment into the lung and development of AHR and airway inflammation.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
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 (T_EFF) 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 T_EFF, 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 T_EFF in the development of allergen-induced AHR and airway inflammation. We show that BLT1-deficient (BLT1^–/–) CD8⁺ T cells or BLT1^–/– T_EFF 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

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Animals

Homozygous CD8^–/– mice, generated by targeting the CD8 -chain gene in C57BL/6 mice (19), OT-1 mice (C57BL/6 strain) expressing a transgenic TCR that is specific for OVA_257–264 (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- TCR, or anti- 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% ⁺CD8⁺ T cells, with <0.5% of cells being ⁺CD8⁺ T cells in preparations from both groups. Contamination by CD4⁺ cells, ⁺ cells, CDllc⁺ cells, or NK1.1⁺ cells in transferred cells was, in total, <0.5%.

Effector CD8⁺ T cell generation

Differentiation of T_EFF 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% /CD8⁺ T cells. CD4⁺ cells, CD11c⁺ cells, NK1.1⁺ cells, or ⁺ cells were <0.1%. The cells were >90% CD8⁺ effector memory cell phenotype, as shown by the CD122^high/CD44^high/CD62L^low cell surface phenotype. T_EFF 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 x 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⁺ T_EFF cells (5 x 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-, 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-.

Cell isolation

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.

Flow cytometry

After purification, 1 x 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- (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.

Statistical analysis

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.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
In vivo-primed CD8⁺ T cells from BLT1^+/+, but not BLT1^–/–, mice fully restore AHR in CD8^–/– mice

Fig. 1A 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 x 10⁶ CD8⁺ T cells from BLT1^+/+ or BLT1^–/– mice were transferred via i.v. injection into the tail vein. As shown in Fig. 1A, 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.

View larger version (28K): [in this window] [in a new window]   FIGURE 1. Reconstitution of CD8–/– mice with in vivo-primed CD8+ T cells from BLT1-deficient mice fails to restore the development of AHR and inflammation. OVA-sensitized CD8–/– mice (recipient mice) received 5 x 106 CD8+ T cells i.v. via the tail vein 2 h before the first airway challenge with aerosolized OVA. Recipient mice were comprised of two groups. In one group, mice received CD8+ T cells from spleens of OVA-sensitized, BLT1-sufficient mice (BLT1+/+ CD8+ T group; n = 8). In the other group, mice received CD8+ T cells from OVA-sensitized, BLT1-deficient mice (BLT1–/– CD8+ T group; n = 8). WT mice and CD8–/– mice receiving no cells are also shown (challenged-only groups: WT C and CD8–/– C; sensitized and challenged groups, WT S/C and CD8–/– S/C; n = 12 in each group). A, AHR was monitored by measuring RL as described in Materials and Methods. B, Cell composition in BAL fluid after transfer of CD8+ T cells. Groups are the same as in A. Total, total cell counts; Mac, macrophages; Lym, lymphocytes; Neu, neutrophils; Eos, eosinophils. C, Levels of IL-13 in BAL fluid after transfer of BLT1–/– CD8+ T cells. Groups are the same as in A. Cytokine levels were measured in supernatants by ELISA, as described in Materials and Methods. D, Numbers of CD8+ T cells in the lung, BAL, PBLN, and spleen in CD8–/– mice after transfer of CD8+ T cells from BLT1+/+ or BLT1–/– mice. Lung, PBLN, and spleen MNC and BAL cells were isolated and stained with anti-CD3 and anti-CD8, and the numbers were calculated as described in Materials and Methods (n = 8 in each group). The results for each group are expressed as the mean ± SEM. *, Significant differences (p < 0.05) are indicated between the BLT1–/– CD8+ T group and the CD8–/– S/C group vs the WT S/C and BLT1+/+ CD8+ T groups. #, Significant differences (p < 0.05) are indicated between the BLT1–/– CD8+ T group and the CD8–/– S/C group vs the WT C and CD8–/– C. **, Significant differences (p < 0.05) are indicated compared with BLT1+/+ CD8+ T.

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. 1B). 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- 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. 1C). 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. 1D 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^–/–, T_EFF fully restores AHR in CD8^–/– mice

We previously showed that transfer of CD8⁺ T_EFF, but not central memory CD8⁺ T cells (T_CM), fully restored AHR and cytokine responses in CD8^–/– recipients (9). To address whether the expression of BLT1 on CD8⁺ T_EFF is required for these airway and cytokine responses, CD8^–/– mice received in vitro-generated BLT1-deficient or BLT1-sufficient CD8⁺ T_EFF cells. Fig. 2A shows that reconstitution of the CD8^–/– mice with T_EFF from BLT1^+/+ mice fully restored AHR to levels comparable to those seen in WT mice. In contrast, transfer of BLT1^–/– T_EFF failed to restore AHR. Transfer of BLT1^–/– T_EFF also failed to restore the number of eosinophils and IL-13 levels in the BAL; transfer of BLT1^+/+ T_EFF fully restored these responses (Fig. 2, B and C).

View larger version (21K): [in this window] [in a new window]   FIGURE 2. Reconstitution of CD8–/– mice with TEFF from BLT1+/+, but not BLT1–/–, mice fully restores the development of AHR and inflammation. CD8–/– recipient mice received 5 x 106 TEFF from BLT1+/+ or BLT1–/– mice i.v. via the tail vein 2 h before the first airway challenge with aerosolized OVA. CD8–/– recipient mice received either BLT1+/+ TEFF (BLT1+/+ TEFF S/C group; n = 8) or BLT1–/– TEFF (BLT1–/– TEFF S/C group; n = 8). Sensitized and challenged WT mice and CD8–/– mice receiving no cells are also shown (WT S/C and CD8–/– S/C; n = 12 in each group). A, AHR was monitored by measuring RL as described in Materials and Methods. B, The numbers of eosinophils in BAL fluid after transfer of BLT1+/+ or BLT1–/– TEFF. Groups are the same as in A. C, IL-13 levels in BAL fluid after transfer of BLT1+/+ or BLT1–/– TEFF. Groups are the same as in A. Cytokine levels were measured in supernatants by ELISA, as described in Materials and Methods. The results for each group are expressed as the mean ± SEM. *, Significant differences (p < 0.05) between BLT1+/+ TEFF recipients after sensitization and challenge (BLT1+/+ TEFF S/C) and WT mice after sensitization and challenge (WT S/C) vs BLT1–/– TEFF recipients after sensitization and challenge (BLT1–/– TEFF S/C) and CD8–/– mice with sensitization and challenge (CD8–/– S/C) group. #, Significant differences (p < 0.05) compared with BLT1+/+ TEFF S/C.

The accumulation of adoptively transferred T_EFF in the lungs of CD8^–/– mice was determined 48 h after the last challenge. Transferred BLT1^+/+ T_EFF cells (identified as CD3⁺CD8⁺) amounted to 11.0 ± 2.5% of the total lung MNC after OVA sensitization and challenge (Fig. 3A), whereas transferred BLT1^–/– T_EFF 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. 3A). More than 95% of the BLT1^+/+ and BLT1^–/– T_EFF in lung and BAL displayed an effector memory phenotype: CD122^high/CD44^high/CD62^low (22). Fig. 3B summarizes the numbers of transferred T_EFF detected in the lungs, PBLN, BAL, and spleen of recipient CD8^–/– mice. Lower numbers of BLT1^–/– T_EFF were detected in BAL and lung, whereas higher numbers of transferred BLT1^–/– T_EFF cells were recovered from PBLN.

View larger version (35K): [in this window] [in a new window]   FIGURE 3. Accumulation of transferred BLT1+/+ and BLT1–/– TEFF in lung and BAL fluid. A, Lung MNC were isolated and stained with anti-CD3 and anti-CD8 Ab as described in Materials and Methods. Quadrants were based on isotype control staining. The data shown are representative of two similar experiments (n = 8 in each group). B, Numbers of CD8+ T cells in lung, BAL, PBLN, and spleen in CD8–/– mice after transfer of BLT1+/+ and BLT1–/– TEFF. Lung, PBLN, and spleen MNC and BAL cells were isolated and stained with anti-CD3 and anti-CD8, and the numbers were calculated as described in Materials and Methods. Groups are the same as in A. *, Significant differences (p < 0.05) compared with BLT1+/+ TEFF group.

In addition to or as a consequence of the marked reduction in recruitment of BLT1^–/– T_EFF, we determined whether the failure to restore AHR and cytokine response by transferred BLT1^–/– T_EFF 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^+/+ T_EFF or BLT1^–/– T_EFF 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. 4A illustrates the percentage of cytokine-producing T_EFF in recipient lungs. IL-13 was detected in 33.3 ± 2.8% of transferred BLT1^+/+ T_EFF cells, whereas among the BLT1^–/– T_EFF 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^–/– T_EFF (5.2 ± 1 vs 4.7 ± 0.9%).

View larger version (26K): [in this window] [in a new window]   FIGURE 4. Intracellular staining of IL-13 in lung CD8+ T cells from recipient CD8–/– mice after transfer of BLT1+/+ and BLT1–/– TEFF. A, Lung MNC were isolated and stimulated with PMA and ionomycin, then cells were fixed, permeabilized, and stained with anti-mouse IL-13 Ab as described in Materials and Methods. CD3+CD8+ cells were gated and analyzed for intracellular detection of IL-13 protein. Quadrants were set based on isotype control staining. The data shown are representative of two similar experiments (n = 8 in each group). B, Percentages of cytokine-producing CD8+ T cells in the lungs of CD8–/– mice after transfer of BLT1+/+ and BLT1–/– TEFF. Groups are the same as in A. C, Absolute numbers of cytokine-producing CD8+ T cells in the lungs of CD8–/– mice after transfer of BLT1+/+ and BLT1–/– TEFF. The numbers were calculated as described in Materials and Methods. Groups are the same as in A. *, Significant differences (p < 0.05) compared with BLT1+/+ TEFF group.

These results demonstrate that in addition to the reduced recruitment, a smaller percentage of the transferred BLT1^–/– T_EFF cells was capable of IL-13 production after airway allergen challenge of sensitized recipients. Overall, the numbers of IL-13-producing BLT1^–/– T_EFF cells were significantly lower than the numbers of IL-13-producing BLT1^+/+ T_EFF cells (Fig. 4, B and C). By contrast, the percentage of IFN--producing cells did not change. These data illustrate that in addition to its role in cell trafficking to the lung, the functional activation of T_EFF and IL-13 production may be dependent in part on the expression of BLT1.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
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 T_EFF cells to sites of inflammation (17, 18, 27, 28). BLT1, the high affinity receptor for LTB₄, has been induced substantially on CD8⁺ T_EFF, with less expression on CD8⁺ T_CM (17). LTB₄ triggers BLT1-dependent chemotaxis of T_EFF, but not naive T cells or T_CM (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⁺ T_EFF, 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⁺ T_EFF 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^+/+ T_EFF 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^–/– T_EFF 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 T_EFF 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^+/+ T_EFF in the lung after transfer were significantly higher than those of BLT1^–/– T_EFF, the numbers of IL-13-producing BLT1^+/+ T_EFF in vitro before transfer were not different from those of BLT1^–/– T_EFF. These data suggest that BLT1^+/+ T_EFF responded in the lung after sensitization and challenge and acquired effector function (IL-13 production), whereas BLT1^–/– T_EFF 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 T_EFF 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.

T_EFF have been reported to produce IFN- in vitro (22, 29). However, after adoptive transfer, T_EFF 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⁺ T_EFF 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, T_EFF 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^–/– T_EFF into lung as well as BAL was significantly impaired compared with that of BLT1^+/+ T_EFF. 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^+/+ T_EFF 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). T_EFF 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⁺ T_EFF into the lung was significantly impaired compared with that of BLT1^+/+ cells; this impairment in the T_EFF 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 T_EFF. These results indicate that although the LTB4-BLT1 pathway regulates CD8⁺ T cell recruitment, it is essential for trafficking of T_EFF 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 T_EFF.

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 FcRI stimulates rapid degranulation and release of LTB4 (43). LTB4 produced by mast cells induces chemotaxis of T_EFF in vitro (27); therefore, after sensitization and challenge with Ag, production of LTB4 from mast cells is probably a major contributor to T_EFF 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 T_EFF 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. TheT_EFF-BLT1 pathway may constitute a novel therapeutic target in bronchial asthma.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Acknowledgments

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

	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 HL36577, HL61005, and AI42246, and by Environmental Protection Agency Grant R835702.

² 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

³ 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; T_CM, central memory CD8⁺ T cell; T_EFF, effector CD8⁺ T cell; WT, wild type.

Received for publication November 17, 2004. Accepted for publication January 18, 2005.

	References

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

1. Busse, W. W., R. F. Lemanske, Jr. 2001. Asthma. N. Engl. J. Med. 344:350.[Free Full Text]
2. Lee, N. A., E. W. Gelfand, J. J. Lee. 2001. Pulmonary T cells and eosinophils: coconspirators or independent triggers of allergic respiratory pathology?. J. Allergy Clin. Immunol. 107:945.[Medline]
3. De Sanctis, G. T., A. Itoh, F. H. Green, S. Qin, T. Kimura, J. K. Grobholz, T. R. Martin, T. Maki, J. M. Drazen. 1997. T-lymphocytes regulate genetically determined airway hyperresponsiveness in mice. Nat. Med. 3:460.[Medline]
4. Robinson, D. S., Q. A. Hamid, S. Ying, A. Tsicopoulos, J. Barkans, A. M. Bentley, C. Corrigan, S. R. Durham, A. B. Kay. 1992. Predominant TH2-like bronchoalveolar T-lymphocyte population in atopic asthma. N. Engl. J. Med. 326:298.[Abstract]
5. Wills-Karp, M., J. Luyimbazi, X. Xu, B. Schofield, T. Y. Neben, C. L. Karp, D. D. Donaldson. 1998. Interleukin-13: central mediator of allergic asthma. Science 282:2258.[Abstract/Free Full Text]
6. Grunig, G., M. Wamock, A. E. Wakil, R. Venkayya, F. Brombacher, D. M. Rennick, D. Sheppard, M. Mohrs, D. D. Donaldson, R. M. Locksley, et al 1998. Requirement for IL-13 independently of IL-4 in experimental asthma. Science 282:2261.[Abstract/Free Full Text]
7. Gonzalez, M. C., P. Diaz, F. R. Galleguillos, P. Ancic, O. Cromwell, A. B. Kay. 1987. Allergen-induced recruitment of bronchoalveolar helper (OKT4) and suppressor (OKT8) T-cells in asthma: relative increases in OKT8 cells in single early responders compared with those in late-phase responders. Am. Rev. Respir. Dis. 136:600.[Medline]
8. Haczku, A., R. Moqbel, M. Jacobson, A. B. Kay, P. J. Barnes, K. F. Chung. 1995. T-cells subsets and activation in bronchial mucosa of sensitized Brown-Norway rats after single allergen exposure. Immunology 85:591.[Medline]
9. Miyahara, N., K. Takeda, T. Kodama, A. Joetham, C. Taube, J. W. Park, S. Miyahara, A. Balhorn, A. Dakhama, E. W. Gelfand. 2004. Contribution of antigen-primed CD8⁺ T cells to the development of airway hyperresponsiveness and inflammation is associated with IL-13. J. Immunol. 172:2549.[Abstract/Free Full Text]
10. Miyahara, N., B. J. Swanson, K. Takeda, C. Taube, S. Miyahara, T. Kodama, A. Dakhama, V. L. Ott, E. W. Gelfand. 2004. Effector CD8⁺ T cells mediate inflammation and airway hyper-responsiveness. Nat. Med. 10:865.[Medline]
11. Samuelsson, B., S. E. Dahlen, J. A. Lindgren, C. A. Rouzer, C. N. Serhan. 1987. Leukotrienes and lipoxins: structures, biosynthesis, and biological effects. Science 237:1171.[Abstract/Free Full Text]
12. Lewis, R. A., K. F. Austen, R. J. Soberman. 1990. Leukotrienes and other products of the 5-lipoxygenase pathway: biochemistry and relation to pathobiology in human diseases. N. Engl. J. Med. 323:645.[Medline]
13. Yokomizo, T., T. Izumi, K. Chang, Y. Takuwa, T. Shimizu. 1997. A G-protein-coupled receptor for leukotriene B4 that mediates chemotaxis. Nature 387:620.[Medline]
14. Kato, K., T. Yokomizo, T. Izumi, T. Shimizu. 2000. Cell-specific transcriptional regulation of human leukotriene B4 receptor gene. J. Exp. Med. 192:413.[Abstract/Free Full Text]
15. Ford-Hutchinson, A. W., M. A. Bray, M. V. Doig, M. E. Shipley, M. J. Smith. 1980. Leukotriene B, a potent chemokinetic and aggregating substance released from polymorphonuclear leukocytes. Nature 286:264.[Medline]
16. Serhan, C. N., S. M. Prescott. 2000. The scent of a phagocyte: Advances on leukotriene B4 receptors. J. Exp. Med. 192:F5.
17. Goodarzi, K., M. Goodarzi, A. M. Tager, A. D. Luster, U. H. von Andrian. 2003. Leukotriene B4 and BLT1 control cytotoxic effector T cell recruitment to inflamed tissues. Nat. Immunol. 4:965.[Medline]
18. Tager, A. M., S. K. Bromley, B. D. Medoff, S. A. Islam, S. D. Bercury, E. B. Friedrich, A. D. Carafone, R. E. Gerszten, A. D. Luster. 2003. Leukotriene B4 receptor BLT1 mediates early effector T cell recruitment. Nat. Immunol. 4:982.[Medline]
19. Hogquist, K., S. Jameson, W. Heath, J. Howard, M. Bevan, F. Carbone. 1994. T cell receptor antagonist peptides induce positive selection. Cell 76:17.[Medline]
20. Fung-Leung, W. P., M. W. Schilham, A. Rahemtulla. 1991. CD8 is needed for development of cytotoxic T cells but not helper T cells. Cell 65:443.[Medline]
21. Tager, A.M., J. H. Dufour, K. Goodarzi, S. D. Bercury, U. H. von Andrian, A. D. Luster. 2000. BLTR mediates leukotriene B4-induced chemotaxis and adhesion and plays a dominant role in eosinophil accumulation in a murine model of peritonitis. J. Exp. Med. 192:439.[Abstract/Free Full Text]
22. Manjunath, N., P. Shankar, J. Wan, W. Weninger, M. A. Crowley, K. Hieshima, T. A. Springer, X. Fan, H. Shen, J. Lieberman, et al 2001. Effector differentiation is not prerequisite for generation of memory cytotoxic T lymphocytes. J. Clin. Invest. 108:871.[Medline]
23. Swanson, B. J., M. Murakami, T. C. Mitchell, J. Kappler, P. Marrack. 2002. RANTES production by memory phenotype T cells is controlled by a posttranscriptional, TCR-dependent process. Immunity 17:605.[Medline]
24. Takeda, K., E. Hamelmann, A. Joetham, L. D. Shultz, G. L. Larsen, C. G. Irvin, E. W. Gelfand. 1997. Development of eosinophilic airway inflammation and airway hyperresponsiveness in mast cell-deficient mice. J. Exp. Med. 186:449.[Abstract/Free Full Text]
25. Tomkinson, A., G. Cieslewicz, C. Duez, K. A. Larson, J. J. Lee, E. W. Gelfand. 2001. Temporal association between airway hyperresponsiveness and airway eosinophilia in ovalbumin-sensitized mice. Am. J. Respir. Crit. Care Med. 163:721.[Abstract/Free Full Text]
26. Oshiba, A., E. Hamelmann, K. Takeda, K. L. Bradley, J. E. Loader, G. L. Larsen, E. W. Gelfand. 1996. Passive transfer of immediate hypersensitivity and airway hyperresponsiveness by allergen-specific immunoglobulin (Ig) E and IgG1 in mice. J. Clin. Invest. 97:1398.[Medline]
27. Ott, V. L., J. C. Cambier, J. Kappler, P. Marrack, B. J. Swanson. 2003. Mast cell-dependent migration of effector CD8⁺ T cells through production of leukotriene B4. Nature Immunol. 4:974.[Medline]
28. Henderson, W. R., Jr, D. B. Lewis, R. K. Albert, Y. Zhang, W. J. Lamm, G. K. Chiang, F. Jones, P. Eriksen, Y. T. Tien, M. Jonas, et al 1996. The importance of leukotrienes in airway inflammation in a mouse model of asthma. J. Exp. Med. 184:1483.[Abstract/Free Full Text]
29. Sallusto, F., D. Lenig, R. Forster, M. Lipp, A. Lanzavecchia. 1999. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 401:708.[Medline]
30. Sad, S., R. Marcotte, T. R. Mosmann. 1995. Cytokine-induced differentiation of precursor mouse CD8⁺ T cells into cytotoxic CD8⁺ T cells secreting Th1 or Th2 cytokines. Immunity 2:271.[Medline]
31. Cerwenka, A., L. L. Carter, J. B. Reome, S. L. Swain, R. W. Dutton. 1998. In vivo persistence of CD8 polarized T cell subsets producing type 1 or type 2 cytokines. J. Immunol. 161:97.[Abstract/Free Full Text]
32. Croft, M., L. Carter, S. L. Swain, R. W. Dutton. 1994. Generation of polarized antigen-specific CD8 effector populations: reciprocal action of interleukin (IL)-4 and IL-12 in promoting type 2 versus type 1 cytokine profiles. J. Exp. Med. 180:1715.[Abstract/Free Full Text]
33. Coyle, A. J., F. Erard, C. Bertrand, S. Walti, H. Pircher, G. Le Gros. 1995. Virus-specific CD8⁺ cells can switch to interleukin 5 production and induce airway eosinophilia. J. Exp. Med. 181:1229.[Abstract/Free Full Text]
34. Seder, R. A., R. Ahmed. 2003. Similarities and differences in CD4⁺ and CD8⁺ effector and memory T cell generation. Nat. Immunol. 4:835.[Medline]
35. Johnson, H. M., B. A. Torres. 1984. Leukotrienes: positive signals for regulation of -interferon production. J. Immunol. 132:413.[Abstract]
36. Farrar, W. L., J. L Humes. 1985. The role of arachidonic acid metabolism in the activities of interleukin 1 and 2. J. Immunol. 1335:1153.
37. Rola-Pleszczynski, M., P. A. Chavaillaz, I. Lemaire. 1986. Stimulation of interleukin 2 and interferon production by leukotriene B4 in human lymphocyte cultures. Prostaglandins Leukotrienes Med. 23:207.[Medline]
38. Arcoleo, F., S. Milano, P. D‘Agostino, E. Cillari. 1995. Effect of exogenous leukotriene B4 (LTB4) on BALB/c mice splenocyte production of Th1 and Th2 lymphokines. Int. J. Immunopharmacol. 17:457.[Medline]
39. Yamaoka, K. A., J. P. Kolb. 1993. Leukotriene B4 induces interleukin 5 generation from human T lymphocytes. Eur. J. Immunol. 23:2392.[Medline]
40. Vargaftig, B. B., M. Singer. 2003. Leukotrienes mediate murine bronchopulmonary hyperreactivity, inflammation, and part of mucosal metaplasia and tissue injury induced by recombinant murine interleukin-13. Am. J. Respir. Cell Mol. Biol. 28:410.[Abstract/Free Full Text]
41. Campbell, J. J., E. C. Butcher. 2000. Chemokines in tissue-specific and microenvironment-specific lymphocyte homing. Curr. Opin. Immunol. 12:336.[Medline]
42. Weninger, W., M. A. Crowley, N. Manjunath, U. H. von Andrian. 2001. Migratory properties of naive, effector, and memory CD8⁺ T cells. J. Exp. Med. 194:953.[Abstract/Free Full Text]
43. Metcalfe, D. D., D. Baram, Y. A. Mekori. 1997. Mast cells. Physiol. Rev. 77:1033.[Abstract/Free Full Text]

This article has been cited by other articles:

				N. Miyahara, H. Ohnishi, H. Matsuda, S. Miyahara, K. Takeda, T. Koya, S. Matsubara, M. Okamoto, A. Dakhama, B. Haribabu, et al. Leukotriene B4 Receptor 1 Expression on Dendritic Cells Is Required for the Development of Th2 Responses and Allergen-Induced Airway Hyperresponsiveness J. Immunol., July 15, 2008; 181(2): 1170 - 1178. [Abstract] [Full Text] [PDF]

				A. Joetham, S. Matsubara, M. Okamoto, K. Takeda, N. Miyahara, A. Dakhama, and E. W. Gelfand Plasticity of Regulatory T Cells: Subversion of Suppressive Function and Conversion to Enhancement of Lung Allergic Responses J. Immunol., June 1, 2008; 180(11): 7117 - 7124. [Abstract] [Full Text] [PDF]

				M. Okamoto, K. Takeda, A. Joetham, H. Ohnishi, H. Matsuda, C. H. Swasey, B. J. Swanson, K. Yasutomo, A. Dakhama, and E. W. Gelfand Essential role of Notch signaling in effector memory CD8+ T cell-mediated airway hyperresponsiveness and inflammation J. Exp. Med., May 12, 2008; 205(5): 1087 - 1097. [Abstract] [Full Text] [PDF]

				W. R. Henderson Jr., E. Y. Chi, J. G. Bollinger, Y.-t. Tien, X. Ye, L. Castelli, Y. P. Rubtsov, A. G. Singer, G. K.S. Chiang, T. Nevalainen, et al. Importance of group X-secreted phospholipase A2 in allergen-induced airway inflammation and remodeling in a mouse asthma model J. Exp. Med., April 16, 2007; 204(4): 865 - 877. [Abstract] [Full Text] [PDF]

				T. Liao, Y. Ke, W.-H. Shao, B. Haribabu, H. J. Kaplan, D. Sun, and H. Shao Blockade of the interaction of leukotriene b4 with its receptor prevents development of autoimmune uveitis. Invest. Ophthalmol. Vis. Sci., April 1, 2006; 47(4): 1543 - 1549. [Abstract] [Full Text] [PDF]

				C. Taube, N. Miyahara, V. Ott, B. Swanson, K. Takeda, J. Loader, L. D. Shultz, A. M. Tager, A. D. Luster, A. Dakhama, et al. The Leukotriene B4 Receptor (BLT1) Is Required for Effector CD8+ T Cell-Mediated, Mast Cell-Dependent Airway Hyperresponsiveness. J. Immunol., March 1, 2006; 176(5): 3157 - 3164. [Abstract] [Full Text] [PDF]

				S. Matsubara, G. Li, K. Takeda, J. E. Loader, P. Pine, E. S. Masuda, N. Miyahara, S. Miyahara, J. J. Lucas, A. Dakhama, et al. Inhibition of Spleen Tyrosine Kinase Prevents Mast Cell Activation and Airway Hyperresponsiveness Am. J. Respir. Crit. Care Med., January 1, 2006; 173(1): 56 - 63. [Abstract] [Full Text] [PDF]

				B. D. Medoff, A. M. Tager, R. Jackobek, T. K. Means, L. Wang, and A. D. Luster Antibody-antigen interaction in the airway drives early granulocyte recruitment through BLT1 Am J Physiol Lung Cell Mol Physiol, January 1, 2006; 290(1): L170 - L178. [Abstract] [Full Text] [PDF]

				R. S. Peebles Jr, K. Hashimoto, J. R. Sheller, M. L. Moore, J. D. Morrow, S. Ji, J. A. Elias, K. Goleniewska, J. O'Neal, D. B. Mitchell, et al. Allergen-Induced Airway Hyperresponsiveness Mediated by Cyclooxygenase Inhibition Is Not Dependent on 5-Lipoxygenase or IL-5, but Is IL-13 Dependent J. Immunol., December 15, 2005; 175(12): 8253 - 8259. [Abstract] [Full Text] [PDF]

				K. Terawaki, T. Yokomizo, T. Nagase, A. Toda, M. Taniguchi, K. Hashizume, T. Yagi, and T. Shimizu Absence of Leukotriene B4 Receptor 1 Confers Resistance to Airway Hyperresponsiveness and Th2-Type Immune Responses J. Immunol., October 1, 2005; 175(7): 4217 - 4225. [Abstract] [Full Text] [PDF]

				T. Okuno, T. Yokomizo, T. Hori, M. Miyano, and T. Shimizu Leukotriene B4 Receptor and the Function of Its Helix 8 J. Biol. Chem., September 16, 2005; 280(37): 32049 - 32052. [Abstract] [Full Text] [PDF]

				N. Miyahara, K. Takeda, S. Miyahara, S. Matsubara, T. Koya, A. Joetham, E. Krishnan, A. Dakhama, B. Haribabu, and E. W. Gelfand Requirement for Leukotriene B4 Receptor 1 in Allergen-induced Airway Hyperresponsiveness Am. J. Respir. Crit. Care Med., July 15, 2005; 172(2): 161 - 167. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Miyahara, N. Articles by Gelfand, E. W.