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,
* Department of Medicine and
Department of Pathology, Harvard Medical School, Boston, MA 02115;
Division of Rheumatology, Immunology, and Allergy and
Department of Pathology, Brigham and Women’s Hospital, Boston, MA 02115; and
¶ Millennium Pharmaceuticals, Cambridge, MA 02139
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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RI activation of ear skin mast cells or zymosan activation of peritoneal macrophages (13). This finding contrasts with the lack of effect of the cys-LTs on the constrictor response of the mouse airways (14, 15). A role for cys-LTs beyond smooth muscle constriction of bronchi and the microvasculature has been suggested in studies showing LTE4-mediated recruitment of eosinophils into asthmatic airways (16) and inhibition of eosinophil and DC influx into airways in human asthma with antagonists for the type 1 cys-LT receptor, the CysLT1R (17, 18). This is supported by the finding that CysLTRs are expressed on hemopoietic cells, such as CD34+ peripheral blood leukocytes, eosinophils, monocytes/macrophages, and mast cells (19, 20). Accumulating evidence in mouse models also suggests a role for 5-LO pathway products in regulating various components of the pulmonary inflammatory response to Ag. In a protocol of OVA sensitization and challenge, 5-LOnull mice with a hybrid C57BL/6 x 129Sv genetic background showed not only reduced airway hyperresponsiveness (AHR) and pulmonary eosinophilic infiltration but also reduced levels of total IgE and Ag-specific IgG in serum compared with wild-type mice (21). When the synthesis of 5-LO products was blocked by the administration of an inhibitor of 5-LO or 5-LO-activating protein (FLAP) to sensitized BALB/c mice before intranasal Ag challenge, pulmonary eosinophilia and goblet cell hyperplasia with mucus plugging were markedly attenuated (22). A subsequent study showing that administration of an antagonist for the CysLT1R had an effect similar to that of the biosynthetic inhibitors implicated cys-LTs as mediators (23). However, the mechanism by which cys-LTs function in this model remained essentially unexplored.
Two recent studies with Ag-sensitized and challenged LTB4R1 (BLT1) null mice with a BALB/c or C57BL/6 background delineated the role of LTB4 in amplifying the effector phase of Th2 cell-dependent pulmonary inflammation. In the BALB/c BLT1Rnull mice, reduced AHR and goblet cell hyperplasia were associated with a deficit in IL-13-producing, but not IL-4- or IL-5-producing, lung CD4+ and CD8+ T cells (24). Adoptive transfer of wild-type T cells into BLT1Rnull mice restored IL-13 levels in the bronchoalveolar lavage (BAL) fluid and normalized the response to OVA. In the C57BL/6 BLT1Rnull mice challenged with OVA, reductions occurred in AHR, goblet cell hyperplasia, tissue eosinophilia, and total IgE, but not in Ag-specific IgG1. Furthermore, when their parabronchial lymph node cells were restimulated with Ag in vitro, their proliferation and generation of IL-5 as well as IL-13 were found to be impaired (25). Thus, both major products of the 5-LO pathway appear to be involved in Ag-induced Th2 cell-dependent pulmonary inflammation.
To define the role of the cys-LTs in Ag-induced pulmonary inflammation, we used a protocol for OVA i.p. sensitization and intranasal challenge that provides a robust elaboration of Ag-specific IgE and IgG1 (26) and induces AHR to methacholine and eosinophilic lung inflammation (27). We found that Ag-induced pulmonary inflammation, characterized by eosinophil infiltration and goblet cell and mast cell hyperplasia, and AHR to methacholine were significantly reduced in the LTC4Snull mice. The production of IL-4, IL-5, and IL-13 by Ag-restimulated parabronchial lymph node cells from sensitized and challenged LTC4Snull mice was also significantly reduced compared with production by wild-type controls, reflecting the Th2 cell dependency of the response. In contrast, there was no suppression of delayed-type hypersensitivity reactions in the skin of LTC4Snull mice. These results identify a role for cys-LTs in the initiation and/or amplification of a pulmonary Th2 response.
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Materials and Methods |
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LTC4Snull mice generated by targeted gene disruption with 129SvEv-derived embryonic stem cells (13) and backcrossed for 6, 8, or 10 generations to a BALB/c background were used. The mouse LTC4S gene is located at chromosome 11B1.1–1.2 (28) 
3.4 mega base pairs centromeric to the Th2 cell cytokine gene cluster and telomeric to the T cell Ig-domain, mucin-domain (Tim) gene family (29). Because a 129SvEv-derived gene segment of chromosome 11 might affect the Th2 cell inflammatory response, we sought to assess the strain purity of the N8 and N9 BALB/c-backcrossed LTC4Snull mice. Microsatellite analysis on chromosome 11 was performed with markers that can differentiate 129Sv and BALB/c strains: D11Mit227, D11Mit134, D11Mit205, D11Mit86, D11Mit23, D11Mit131, D11Mit274, D11Mit242, D11Mit41, D11Mit67, D11Mit337, and D11Mit48 (Charles River Laboratories). We found that the Th2 cell cytokine gene cluster and chemokine gene cluster were derived from BALB/c (data not shown).
The Tim gene family, particularly Tim1, contains polymorphisms that may affect the development of AHR and T cell production of IL-4 and IL-13 after Ag sensitization and challenge (29). Tim 1, which is predominantly expressed on Th2 cells, has 3-aa substitutions and 15-aa deletions in the DBA/2 and C57BL/6 strains relative to the BALB/c strain. Because the microsatellite analysis could not distinguish Tim1 gene polymorphisms between the 129Sv and BALB/c strains, we cloned and sequenced exons 2 and 3 of the Tim1 gene from six independent LTC4Snull mice of the N8 generation. PCR was performed with tail DNA from each mouse and primers (exon 2 sense primer, 5'-TGTTGAATACTAATTCCCCA-3'; exon 2 antisense primer, 5'-CTGGTTTAACTTGCAATGAA-3'; exon 3 sense primer, 5'-CGAAGCTCTTGTCACTTCTT-3'; exon 3 antisense primer, 5'-TTATTTGGGAGAACTGTCTG-3'). We found that the Tim1 sequences from all six LTC4Snull mice were the same as those for the BALB/c Tim1 gene.
Sensitization and challenge with OVA
Eight- to 12-wk-old female LTC4Snull mice and wild-type littermates (N6 and N8) or age-matched female BALB/c mice (Charles River Laboratories) were immunized as described (26, 27). Mice received an i.p. injection of 10 µg of OVA (Grade V; Sigma-Aldrich) dissolved in 100 µl of endotoxin-free saline or 100 µl of saline alone for controls on days 0, 2, 4, 6, 8, 10, and 12. On days 40, 43, and 46, mice were challenged with either 20 µg of OVA in 20 µl of saline or 20 µl of saline alone by intranasal droplet application under light anesthesia with isoflurane. All animal studies were approved by the Animal Care and Use Committee of the Dana-Farber Cancer Institute.
Histology and histochemistry
Mice were killed by i.p. injection of an overdose of pentobarbital 48 h after the last challenge. The left lungs and the tracheas were fixed for at least 8 h in 4% paraformaldehyde. Some portions of this tissue were embedded in paraffin and others in glycolmethacrylate, as described previously (30). Four-micrometer-thick paraffin sections were stained with H&E for general morphologic examination. Two-micrometer-thick glycolmethacrylate sections were stained by the chloroacetate esterase reaction to identify mast cells and neutrophils and counterstained with hematoxylin. When three or more of their secretory granules were found outside the cell, the mast cell was counted as degranulating (activated) (31). These plastic preparations were used for morphometry. Other plastic sections were stained with Congo red to identify eosinophils or with Jones’ methenamine silver procedure (32) to color mucus, goblet cells, and extracellular matrix proteins black.
The extent of cellular infiltration in the bronchovascular bundles was quantitated without knowledge of the particular mouse strain or procedure as previously described for bleomycin-induced lung injury (33, 34, 35). Ten randomly selected areas of each lung were photographed at low magnification (x10), and the areas of extensive cellular infiltration were outlined. After conversion of the photographs to digital images with a flatbed computer scanner (UMAX Power Look III) and software (Adobe Photoshop 7.0), the number of pixels contained in the areas with cellular infiltration and the number of pixels contained in the image of the entire lung field were determined with histogram function. The total number of pixels outlined as cellular infiltrate was divided by the total number of pixels in the entire lung field and multiplied by 100 to generate a percentage of area with cellular infiltration for each mouse.
Measurement of total and OVA-specific serum Igs
Sham and OVA-sensitized mice that had been challenged intranasally with OVA or saline were killed with an i.p. injection of an overdose of pentobarbital, and cardiac puncture was performed to obtain serum for measurement of Igs. Total IgE, IgG1, and IgG2a were determined with ELISA kits (IgE and IgG2a: BD Pharmingen; IgG1: Assay Designs). OVA-specific IgE and IgG2a were measured in the following manner. 96-well plates were coated with either anti-mouse IgE or IgG2a Ab (2 µg/ml) and incubated at 4°C overnight. The plates were washed and nonspecific binding was blocked with PBS containing 1% heat-inactivated FCS. Serially diluted sera were added, and the plates were incubated for 2 h at room temperature. Biotinylated OVA (2 µg/ml) was then added to each well, and the plates were incubated with streptavidin-conjugated HRP for 1 h. The plates were washed multiple times with PBS with 0.05% Tween 20, substrate solution (tetramethylbenzidine and hydrogen peroxide) was added, and the plates were incubated for 30 min in the dark. The reaction was terminated by the addition of 1 M H2SO4. Absorbance was read at 450 nm and 570 nm, and absorbance at 570 nm was subtracted from absorbance at 450 nm. OVA-specific Ab concentrations were calculated by comparison to standards: monoclonal anti-OVA IgE (provided by Dr. L. Kobzik, Harvard School of Public Health, Boston, MA) (36), and pooled hyperimmune serum with high-titer anti-OVA IgG2a (concentration arbitrarily set at 30,000 U/ml) generated by repeated i.p. injection of BALB/c mice with OVA with alum (37). For determination of OVA-specific IgG1, the plate was coated with OVA (10 µg/ml), the serially diluted sera were added, and the plate was incubated with biotinylated anti-mouse IgG1 (BD Pharmingen) and streptavidin-conjugated HRP. Anti-OVA mouse IgG1 mAb (clone OVA-14; Sigma-Aldrich) was used as the standard.
Measurement of cytokine mRNA expression in the lung
Total RNA was isolated from the right lungs by homogenization in RNA Stat-60 (Tel-Test) according to the manufacturer’s instructions. Samples were treated with DNase I (12 U/ml; Qiagen) for 15 min at room temperature and purified by using the RNAeasy minikit (Qiagen). Quantities of mRNA for IL-4, IL-5, IL-13, eotaxin, IL-10, IFN-
, IL-12 p35, and IL-12 p40 were measured relative to GAPDH using the TaqMan system (Applied Biosystems) with gene-specific primers.
Cytokine production by parabronchial lymph node cells after in vitro restimulation with OVA
LTC4Snull mice and their littermates (N8 generation, BALB/c) were immunized and challenged as above. Forty-eight hours after the last intranasal challenge, the parabronchial lymph nodes were excised from each mouse and homogenized. The cell suspensions were filtered through a 70-µm cell strainer, centrifuged at 300 x g for 5 min at room temperature, and resuspended in RPMI 1640 medium containing 10% FCS. After the total number of cells was counted for each mouse, 2 x 106 cells were cultured in a 24-well plate in the presence of 0.1 or 1 mg/ml OVA for 72 h. The concentrations of IL-4, IL-5, IL-13, and IFN- in the supernatants were measured with ELISA kits (R&D Systems).
Delayed-type hypersensitivity assays
LTC4Snull mice (N10) and wild-type littermates were injected s.c. at the tail base with 100 µg OVA emulsified in CFA. After 6 days, mice were challenged with 50 µg of OVA in one hind footpad and saline in the other hind footpad. Footpad thickness was measured with a dial thickness gauge (Dyer) 24 h after challenge. Specific swelling was determined by subtracting nonspecific swelling in the saline-injected foot from that in the Ag-injected foot. For a contact hypersensitivity assay, LTC4Snull mice (N10) and wild-type littermates were sensitized by the application of 100 µl of 0.5% FITC (Sigma-Aldrich; dissolved in 1:1 acetone/dibutyl phthalate) to the abdominal skin. After 6 days, a contact hypersensitivity reaction was elicited by the topical application of 20 µl of 0.5% FITC to the right ear of each mouse. The left ear of each mouse received vehicle alone. Twenty-four hours later, mice were killed and a 6-mm-diameter disc of tissue was obtained from the center of each ear and weighed. The difference in wet weight between experimental (right) and control (left) ears was calculated in mice of each genotype.
Measurement of AHR
LTC4Snull mice and their littermates (N6 and N8 generation, BALB/c) were immunized and challenged as above. Twenty-four hours after the last challenge, mouse AHR to aerosolized methacholine was measured with a whole body plethysmograph (Buxco Electronics) as described previously (38). Unrestrained conscious mice were placed in whole body plethysmographic chambers. After a 5-min stabilization period, increasing concentrations of aerosolized methacholine (0, 2.5, 5, 10, 20, 30, and 40 mg/ml in saline) were introduced for 2 min each, and mean airway bronchoconstriction readings, as assessed by enhanced respiratory pause (Penh), were obtained over a 5-min period after the administration of each concentration.
In vitro Th cell proliferation and differentiation, Ag presentation, and B cell function
CD4+ T cells were purified from spleen and lymph nodes (cervical, axillary, inguinal, and mesenteric) of LTC4Snull and BALB/c mice by positive selection with MACS CD4 microbeads (Miltenyi Biotec) according to the manufacturer’s instructions. T cells were cultured in RPMI 1640 medium containing 10% FCS at a concentration of 1 x 105 cells/well on anti-CD3 mAb-coated plates (1 µg/ml, clone 2C11; BD Pharmingen) with and without anti-CD28 mAb (4 µg/ml, clone 37.51; BD Pharmingen) for 72 h at 37°C. [3H]Thymidine incorporation was measured 18 h after the addition of 1 µCi of [3H]thymidine. In separate anti-CD3 mAb-coated plates, Th2 cell differentiation was promoted in the presence of IL-4 (20 ng/ml; PeproTech) and anti-IFN- mAb (20 µg/ml, clone XMG1.2; BD Pharmingen), and Th1 cell differentiation was promoted by the addition of IL-12 (6 ng/ml; PeproTech) and anti-IL-4 mAb (20 µg/ml, clone 11B11; National Institutes of Health). No cytokine or anti-cytokine Ab was added for the Th0 condition. Recombinant human IL-2 (100 U/ml; National Institutes of Health) was added the next day, and cells were cultured for 5 days. T cells were stimulated with PMA (50 ng/ml) and ionomycin (1 µM) for 4 h at 37°C. Cytokine secretion was blocked by incubation with monensin (3 µM) for 2 h. Cells were fixed with 2% paraformaldehyde in PBS for 20 min at room temperature, and nonspecific binding was blocked by incubation with mouse IgG (100 µg/ml; Sigma-Aldrich) in PBS containing 0.5% FCS, 0.05% sodium azide, and 0.5% saponin for 10 min at 4°C. FITC- or PE-conjugated anti-mouse IL-4, mouse IL-5, mouse IL-10, mouse IFN-, mouse IL-2, and mouse TNF-
Abs were used. For staining with mouse IL-13, biotin-conjugated anti-mouse IL-13 Ab and streptavidin conjugated with FITC were used. Cells were analyzed on a BD Biosciences FACScan with the CellQuest software, and the cells containing cytokines were shown as a percentage of total cells.
For assays of APC function, CD4+ T cells were isolated from female DO11.10 mice (BALB/c background; The Jackson Laboratory) that express the TCR specific for an OVA323–339 peptide by positive selection with MACS CD4 microbeads. APCs were prepared from the spleens of LTC4Snull and BALB/c mice by homogenizing the tissues, lysing the RBCs, and irradiating the splenocytes with 2500 rad. DO11.10 CD4+ T cells were plated in 24-well plates at 2.5 x 105 cells/well in the presence or absence of the OVA323–339 peptide (0.3 µM) at 37°C. APCs were added at 5 x 106 cells/well. [3H]Thymidine incorporation, Th cell differentiation, and intracellular cytokine staining were evaluated as described above.
To induce in vitro IgG1 and IgE production, splenocytes (5 x 105 cells/well) were cultured for 5 days with isotype-matched control mAb (hamster IgM; BD Pharmingen) alone or with IL-4 (50 ng/ml), or with IL-4 plus anti-CD40 mAb (1 µg/ml, clone HM40–3; BD Pharmingen).
Statistical analysis
Results were expressed as means ± SEM. The Student t test was used for the statistical analysis in cases in which the variance was homogeneous, and Welch’s test was used when the variance was heterogeneous. A value of p < 0.05 was considered significant.
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Results |
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To examine the role of cys-LTs in Ag-induced pulmonary inflammatory responses, we examined lung tissue histologically 48 h after the last challenge of sensitized LTC4Snull and wild-type mice. Lungs of saline-treated wild-type mice and LTC4Snull mice were not inflamed. In the lungs of saline-treated wild-type mice, bronchi and their associated blood vessels (bronchovascular bundles) were prominent throughout (Fig. 1a). Small aggregates of lymphocytes, but not eosinophils, were present occasionally in association with the bronchi in saline-treated mice (Fig. 1d). The bronchovascular bundles were surrounded by basement membranes and a delicate meshwork of collagen, both of which stained black with methenamine silver (Fig. 1g). Goblet cells were methenamine silver-negative (Fig. 1g).
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To quantitate the extent of pulmonary inflammation, we compared the ratios of inflamed area to total lung area in OVA-treated LTC4Snull mice and wild-type mice. Ten low power (x10 objective) fields of randomly selected areas were examined for digital image analysis. After OVA challenge, the LTC4Snull mice had inflammatory infiltrates occupying 3% of the lung, whereas the wild-type mice had lesions occupying 13% (Fig. 2a).
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cys-LTs play a role in regulating Th2 cell-dependent immune responses
Baseline and elicited Ig response. To assess the role of cys-LTs in the Ag-specific Ab response, serum was collected from wild-type and LTC4Snull mice by cardiac puncture 48 h after the last challenge with saline or OVA, and total and OVA-specific IgE, IgG1, and IgG2a were measured by ELISA. After sensitization and challenge with OVA, LTC4Snull mice had significantly less OVA-specific IgE and OVA-specific IgG1, but comparable levels of OVA-specific IgG2a as compared with the wild-type mice (Fig. 3). OVA-specific IgE accounted for virtually all of the total IgE in the OVA-treated mice and OVA-specific IgG1 accounted for the increment in total IgG1 in the OVA-treated mice, as compared with saline-treated controls. Saline-treated LTC4Snull mice had significantly less total IgE than wild-type mice, but had comparable levels of total IgG1 and IgG2a (Fig. 3). The reduced levels of total IgE in serum were confirmed with N10-backcrossed LTC4Snull mice, which had a chromosome 11 of complete BALB/c origin by microsatellite analysis (data not shown).
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, IL-12 p35, and IL-12 p40 (Fig. 4). OVA-treated LTC4Snull mice had significantly reduced expression of IL-5, IL-13, eotaxin, and IL-10 as compared with wild-type mice (Fig. 4).
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in the supernatants were measured by ELISA. The total number of lymph node cells recovered from the LTC4Snull mice was significantly less than the number of cells recovered from the wild-type mice (1.15 ± 0.16 x 107 cells/mouse vs 1.76 ± 0.10 x 107 cells/mouse, n = 5, p < 0.05). When compared on a per cell basis using 2 x 106 lymph node cells, the levels of IL-4, IL-5, and IFN- were significantly reduced in the LTC4Snull mice after in vitro restimulation with 0.1 mg/ml OVA, and the level of IL-13 was significantly reduced after restimulation with both 0.1 and 1 mg/ml OVA in the LTC4Snull mice as compared with wild-type controls (Fig. 5).
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Because IFN- production by restimulated parabronchial lymph node cells from OVA-treated LTC4Snull mice was also reduced compared with that of wild-type controls, we examined a possible role for the cys-LTs in integrated Th1 cell-mediated inflammatory responses with the BALB/c-backcrossed (N10) LTC4Snull mice. For a classical delayed-type hypersensitivity response, we immunized wild-type and LTC4Snull mice with OVA plus CFA, challenged the footpads with OVA or saline at day 6, and compared footpad swelling 24 h after challenge. There was no difference in specific swelling between wild-type and LTC4Snull mice (Fig. 6a). For a contact hypersensitivity reaction, wild-type and LTC4Snull mice were sensitized by application of 0.5% FITC to the abdominal skin and challenged 6 days later by the topical administration of FITC or vehicle to the ears; tissue swelling in the experimental and the control ears was compared 24 h after challenge (42). There was no difference in ear wet weight change between wild-type and LTC4Snull mice (Fig. 6b).
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As a physiologic parameter of lung function, AHR to aerosolized methacholine was assessed by monitoring the Penh, a parameter that correlates with airway conductance in the BALB/c strain (38, 43), 24 h after the last intranasal challenge. The LTC4Snull mice (N6 and N8) were significantly less responsive to concentrations of aerosolized methacholine ranging from 5 to 40 mg/ml than the OVA-sensitized and challenged wild-type mice (Fig. 7). AHR to methacholine in mice sensitized and challenged with saline was the same for the LTC4Snull and wild-type mice at all concentrations of methacholine tested.
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To address the mechanism by which the Th2 cell cytokine response in the lung is impaired in LTC4Snull mice after OVA sensitization and intranasal challenge, we compared the capacities of naive CD4+ T cells from LTC4Snull and wild-type mice to proliferate and differentiate into Th1 and Th2 cells in vitro. [3H]Thymidine incorporation by CD4+ T cells isolated from LTC4Snull and wild-type mice in response to either anti-CD3 or anti-CD3 plus anti-CD28 was essentially the same (data not shown). Intracellular cytokine staining of CD4+ T cells maintained as Th0 or polarized in vitro toward Th1 and Th2 showed essentially the same percentage of positive cells in the two genotypes for each condition (data not shown).
Because no intrinsic T cell defect was recognized, we examined whether APCs from LTC4Snull mice are capable of supporting proliferation and differentiation of CD4+ T cells toward the Th1 and Th2 phenotypes. Irradiated splenocytes from naive LTC4Snull and wild-type mice were incubated with CD4+ T cells isolated from OVA-specific TCR transgenic DO11.10 mice in various concentrations of an OVA peptide. The dose response to peptide presentation by the splenocytes from LTC4Snull mice was indistinguishable from that from wild-type mice as assessed by [3H]thymidine incorporation (data not shown). Intracellular cytokine staining showed that APCs from LTC4Snull mice could support in vitro Th cell differentiation toward Th1 and Th2 similarly to APCs from wild-type mice (data not shown).
To determine whether there was an intrinsic defect in IL-4-dependent class switching of B cells from the LTC4Snull mice, we cultured splenocytes in the presence of IL-4 with or without anti-CD40 mAb for 5 days and measured total IgE and IgG1 in the supernatants. Culture of the cells with IL-4 and anti-CD40 mAb substantially augmented IgE and IgG1 production in vitro, and there was no difference in the Th2 cell-associated Ig production between splenocytes from wild-type and LTC4Snull mice (data not shown).
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Discussion |
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IL-4 regulates the levels of total serum IgE and of Ag-specific IgE and IgG1 (37, 44) and is needed for Th2 cell polarization and the generation of downstream effector Th2 cytokines (37, 45), such as IL-5 and eotaxin for full pulmonary eosinophilia (44, 45), IL-13 for induced AHR and goblet cell hyperplasia (46, 47, 48), and IL-9 for expansion of the intraepithelial mast cell population (49). Each of these manifestations of a Th2 cell-dependent pulmonary inflammatory response was diminished in the LTC4Snull mice. The morphologic manifestations of the Th2 cell-dependent pulmonary inflammation, including accumulation of eosinophils and lymphocytes in bronchovascular bundles, goblet cell hyperplasia with mucus production, and thickening of the bronchial basement membrane were markedly reduced in the LTC4Snull mice as compared with their controls (Fig. 1). Quantitation of the reduced cellular infiltration of the bronchovascular bundles by digital image analysis confirmed that it was significant (Fig. 2a). An impaired Th2 cell response would account for the deficiency in intraepithelial mast cells (Figs. 1l and 2b), and the significantly reduced levels of Ag-specific IgE and IgG1 (Fig. 3) would limit activation of those mast cells present. Finally, a functional readout of an Ag-induced airway response, AHR to methacholine challenge, was significantly attenuated in the LTC4Snull mice as compared with their controls (Fig. 7).
This profound impairment of a Th2 cell-dependent phenotype in LTC4Snull mice was associated with decreased levels of transcripts for IL-5, IL-13, eotaxin, and IL-10 in lungs of Ag-treated LTC4Snull mice compared with Ag-treated wild-type mice (Fig. 4). Furthermore, in vitro Ag restimulation of parabronchial lymph node cells revealed a significant loss in IL-4, IL-5, and IL-13 generation on a per cell basis (Fig. 5) that would be compounded on a per mouse basis by the reduced number of cells. Although IL-5 and IL-13 levels from sensitized and challenged LTC4Snull mice were reduced, whether assessed by transcript expression in total lung or by protein production from ex vivo Ag restimulation of parabronchial lymph node cells, there was a discrepancy for IL-4, which was reduced only in the restimulation assay. In a preliminary experiment, in which we isolated lung mononuclear cells 2 days after the last Ag challenge and performed intracellular cytokine staining after activation with PMA and ionomycin (24), we observed a marked reduction in number of CD4+ T cells expressing IL-4, IL-5, IL-10, and IL-13 from the Ag-sensitized and challenged LTC4Snull mice relative to controls (data not shown). The sustained level of IL-4 transcripts in the lung of sensitized and challenged LTC4Snull mice may reflect a contribution by activated eosinophils, basophils, mast cells, and/or NKT cells, as well as different kinetics for cellular recruitment to the lung and for transcription, translation, and release of cytokines, and will be addressed in future studies.
As our studies were progressing, two laboratories reported that in mice lacking the BLT1R, a pulmonary inflammatory response induced by i.p. sensitization with adjuvant and aerosol challenge of Ag was impaired (24, 25). Because of the profound effect of the strain background, Ag-induced pulmonary inflammation is more robust in the BALB/c than in the C57BL/6 strain (29, 50). Thus, it seemed reasonable to compare our findings with the LTC4Snull mice with those for the BLT1Rnull mice with a BALB/c background (24). The BLT1Rnull mice showed a reduction in the number of IL-13-expressing, but not IL-4- or IL-5-expressing lung CD4+ and CD8+ T cells and a reduction in IL-13 generation from lung mononuclear cells, but not from parabronchial lymph node cells restimulated with Ag in vitro. There was an associated loss in Ag-induced AHR, goblet cell hyperplasia, and content of IL-13 in the BAL fluid of the BALB/c BLT1Rnull mice. In contrast, the LTC4Snull mice had a more global defect involving steady-state transcription of Th2 cell cytokines in the lung (Fig. 4) and generation of cytokines by Ag-restimulated parabronchial lymph node cells that included IL-4, IL-5, and IL-13 (Fig. 5). It thus seems likely that the contributions of LTB4 and the cys-LTs are not redundant to the development of the Th2 cell-dependent pulmonary inflammatory response of the BALB/c strain. Inasmuch as exogenously added LTB4 and LTC4 both can augment the phagocytosis of opsonized Klebsiella by alveolar macrophages in the innate host defense (51), they may have another type of integrated proinflammatory function in allergic disease.
There are several possible mechanisms for the occurrence of such a profound phenotype in the BALB/c LTC4Snull mice involving migration and/or function of DCs or responding T cells or both. The cys-LTs not only constrict microvascular smooth muscle and cause plasma leakage but also increase the expression of adhesion molecules, such as P-selectin, on endothelial cells (52). The migration of FITC-bearing skin DCs to regional lymph nodes is impaired in mice lacking multidrug resistance-associated protein 1, which can transport LTC4 (53). In a study directed to DC function in lung, mouse bone marrow-derived DCs pulsed with mite Ag alone, with Ag plus LTD4, or with Ag plus an antagonist for the CysLT1R were intranasally injected into BALB/c recipients who were then intranasally challenged with Ag. The percentage of eosinophils and the concentration of IL-5 in BAL fluid were increased in mice injected with DCs pulsed with Ag plus LTD4 and were decreased in mice injected with DCs pulsed with Ag plus a CysLT1R antagonist as compared with mice injected with DCs pulsed with Ag alone (54). The concentration of IFN- in BAL fluid was increased in mice injected with DCs pulsed with Ag plus a CysLT1R antagonist (54). In another pharmacologic study, the treatment of BALB/c mice with CysLT1R antagonists during OVA sensitization reduced the production of IL-4, IL-5, and IFN- in BAL fluid without affecting the levels of OVA-specific IgE and IgG in serum (55). Lung DCs harvested from OVA-sensitized and aerosol-challenged mice treated with a CysLT1R antagonist during sensitization supported less OVA-specific CD4+ T cell proliferation. Similarly, splenic DCs harvested from OVA-sensitized mice treated with a CysLT1R antagonist during sensitization supported less OVA-specific CD4+ T cell proliferation and produced less IL-10 and IL-12 in response to LPS as compared with DCs from OVA-sensitized mice not treated with a CysLT1R antagonist (55). Finally, in a Th1 cell-biased immunization protocol, splenic DCs from mice sensitized with OVA plus CFA and treated with a CysLT1R antagonist also produced less IL-10 and IL-12 in response to LPS (55). Thus, cys-LTs may augment DC function in a Th2-dominant manner or in both Th2- and Th1-biased immune responses in some conditions.
The studies implicating cys-LT-augmented DC migration and/or function in the in vivo response to Ag sensitization and/or challenge do not exclude an action on T cells. FTY720, a sphingosine-1-phosphate receptor ligand, promotes T cell migration from spleen and peripheral blood to lymph nodes, and the FTY720-mediated T cell migration was diminished in mice null for multidrug resistance-associated protein 1 or 5-LO, suggesting a role of cys-LTs in T cell migration (56). Recently, it was shown that mouse CD4+ and CD8+ T cells activated by mAbs to CD3 and CD28 can migrate in response to LTD4 in vitro and this migration is blocked by a CysLT1R antagonist, MK-571 (57). The chemotactic response of the T cells could also be a surrogate for other CysLTR-mediated effects. For example, cord blood-derived human mast cells respond to cys-LTs with induced expression of transcripts and protein for various cytokines and chemokines such as IL-5, IL-8, and TNF- (58, 59). The observation that CysLT1R mRNA expression is highly up-regulated in CD4+ T cells cultured in either Th1- or Th2-polarizing conditions (57) prompted us to consider whether a Th1 cell-mediated immune response is also impaired in the absence of cys-LTs. The delayed-type hypersensitivity response to Ag sensitization with CFA and footpad challenge was comparable between BALB/c-backcrossed LTC4Snull mice and their wild-type mice (Fig. 6a). Cutaneous contact hypersensitivity to FITC was also not different between the LTC4Snull mice and wild-type controls (Fig. 6b). These findings are compatible with earlier studies showing that contact hypersensitivity responses induced by FITC administration are normal in 5-LOnull mice and FLAPnull mice on 129Sv genetic background compared with wild-type mice (42). Thus, it appears that the role of the 5-LO pathway, and particularly the role of the LTC4S-derived cys-LTs, in Ag-induced inflammation is to preferentially effect a Th2 cell-type response and its histologic signatures. Alternatively, the intact delayed-type and contact hypersensitivity reactions in the skin of the LTC4Snull mice may reflect a tissue-specific immunologic difference and does not definitively limit cys-LT function to allergic Th2 responses. To directly address the specific role of the cys-LTs in Th1 vs Th2 pulmonary inflammation, we will study the immunologic response of LTC4Snull mice with BALB/c and C57BL/6 backgrounds that are sensitized with Ag and either high- or low-dose LPS to direct the response toward Th1 or Th2 type inflammation, respectively (60, 61). Nevertheless, our findings extend the possible role of the cys-LTs beyond smooth muscle constriction to the adaptive immune component of the inflammatory cell response.
The findings by others that the transcripts for the CysLT1R, 5-LO, and FLAP are up-regulated in DCs by an Ag pulse (54) and that the CysLT1R is induced at the transcript and functional levels by the activation of T cells (57) suggest that cys-LTs could bifunctionally modulate the DC/T cell interaction. The fact that immune response stimulation is needed for CysLT1R expression in both cell types could account for the profound in vivo phenotype of the LTC4Snull mice in contradistinction to the normal responses of these cell types from naive mice studied in vitro. In a possible schema, Ag-stimulated DCs would elaborate and respond to cys-LTs through induced CysLT1R expression with migration to regional lymph nodes. The resulting activation of T cells with CysLT1R expression could lead to migration toward lung tissue sources of cys-LTs, such as eosinophils or mononuclear cells, in bronchovascular bundles so as to supply Th2 cell cytokines in a pulmonary setting. This scheme argues that the primary effect of cys-LTs is on DCs with an amplification role for T cells. Indeed, bone marrow-derived DCs from LTC4Snull and CysLT1Rnull mice with a C57BL/6 background had a significant defect in in vitro chemotaxis to CCL19, a CCR7 ligand (G. J. Randolph, Mt. Sinai School of Medicine, unpublished observation), and DC migration from the airways to the draining lymph nodes in response to intratracheally injected FITC-OVA was significantly reduced in naive BALB/c LTC4Snull mice as compared with wild-type controls (data not shown). Future studies with adoptive transfer of wild-type T cells into compound Rag2null, LTC4Snull mice, and with transfer of OVA-specific TCR transgenic T cells with a null mutation of the CysLT1R into naive wild-type mice will be required to assess the contribution of cys-LTs to DC and T cell functions, respectively. Nonetheless, the phenotypic characterization of LTC4Snull mice in the present study implies a unique and broader role for the cys-LTs in the development and amplification of the Th2 cell-dependent response in the lung.
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1 This work was supported by National Institutes of Health Grants AI-07306, AI-31599, HL-36110, and AI-52353. F.I.H. is funded by a grant from the American Academy of Allergy, Asthma, and Immunology. 
2 D.C.K. and F.I.H. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Yoshihide Kanaoka, Brigham and Women’s Hospital, Smith Building, Room 626C, One Jimmy Fund Way, Boston, MA 02115. E-mail address: ykanaoka{at}rics.bwh.harvard.edu
4 Abbreviations used in this paper: LT, leukotriene; 5-LO, 5-lipoxygenase; cys-LT, cysteinyl leukotriene; LTC4S, LTC4 synthase; AHR, airway hyperresponsiveness; FLAP, 5-LO-activating protein; CysLT1R, type 1 cys-LT receptor; BLT1R, type 1 LTB4 receptor; BAL, bronchoalveolar lavage; Penh, enhanced pause; Tim, T cell Ig-domain, mucin-domain.
Received for publication November 1, 2005. Accepted for publication January 23, 2006.
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, ELC)-dependent mobilization of dendritic cells to lymph nodes. Cell 103: 757-768. [Medline]- and 
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; n = 8 for saline (SAL)-treated group and n = 16 for OVA-treated group) and LTC4Snull mice (
; n = 8 for saline (SAL)-treated group and n = 17 for OVA-treated group) were outlined and quantitated by image analysis as described in Materials and Methods. b, The total and degranulated/activated (Act) mast cells (MC) in epithelium (Epi) and submucosa (Sub) in a tracheal section of wild-type mice (
and