Abstract
Rationale: Allergic asthma is characterized by reversible airway obstruction, lung inflammation, and airway hyperresponsiveness (AHR). Previous studies using leukotriene B4 (LTB4) receptor 1–deficient mice and adoptive transfer experiments have suggested that LTB4 plays a role in lung inflammation and AHR.
Objectives: In this study, we used a leukotriene A4 hydrolase (LTA4H) inhibitor as a pharmacological tool to directly examine the role of LTB4 in a mast cell–dependent murine model of allergic airway inflammation.
Methods: We used the forced oscillation technique to test the effects of an LTA4H inhibitor dosed during the challenge phase on AHR. Lung tissue and lavage were collected for analysis.
Measurements and Main Results: Treatment with an LTA4H inhibitor improved multiple parameters encompassing AHR and lung function. Significant decreases in inflammatory leukocytes, cytokines, and mucin were observed in the lung lumen. Serum levels of antigen-specific IgE and IgG1 were also decreased. Labeled antigen uptake by lung dendritic cells and subsequent trafficking to draining lymph nodes and the lung were decreased on LTA4H inhibitor treatment. Provocatively, inhibition of LTA4H increased lipoxin A4 levels in lung lavage fluid.
Conclusions: These data suggest that LTB4 plays a key role in driving lung inflammation and AHR. Mechanistically, we provide evidence that inhibition of LTA4H, affects recruitment of both CD4+ and CD8+ T cells, as well as trafficking of dendritic cells to draining lymph nodes, and may beneficially modulate other pro- and antiinflammatory eicosanoids in the lung. Inhibition of LTA4H is thus a potential therapeutic strategy that could modulate key aspects of asthma.
Leukotriene B4 (LTB4) is a key inflammatory mediator capable of attracting and activating multiple cell types. It is increased in allergic airway disease but its exact role in processes leading to airway hyperreactivity is not well defined.
Pharmacological inhibition of leukotriene A4 hydrolase decreases LTB4 in the airways in a mouse asthma model and attenuates airway inflammation and airway hyperreactivity through modulation of T cell and dendritic cell function.
Allergic asthma is characterized by reversible airway obstruction, lung inflammation, and AHR. Elevated levels of LTB4 have been reported in a number of inflammatory disorders, including sputum and bronchoalveolar lavage (BAL) samples from patients with chronic obstructive pulmonary disease, allergy, and asthma (8–10). In addition, LTA4H levels are increased in circulating neutrophils and the airways of patients with asthma (11, 12). Evaluation of lung inflammation in BLT1-deficient mice on either the Balb/C or C57/B6 genetic background has yielded conflicting results. In Balb/C mice, no changes were seen in serum IgE or airway eosinophilia, whereas BLT1-deficient B6 mice showed a dramatic decrease in both parameters (13, 14). However, both studies reported attenuated AHR as well as decreased levels of IL-13 in BAL fluid. More recently mast cells were identified as the main source of LTB4 that control the recruitment of IL-13–producing T cells (15).
Clinically, inhibition of cysteinyl leukotriene (CysLT) activity by agents such as montelukast has proved an effective treatment for various aspects of asthma, at least in a portion of patients (16), whereas attenuation of both CysLT and LTB4 production through the upstream inhibition of 5-LO have suggested additional clinical benefit through this approach (17). In addition, genetic polymorphisms in both the 5-LO and LTA4H gene are associated with asthma susceptibility (18) and variation in response to montelukast therapy (19), suggesting potential clinical usefulness for LTB4 inhibition.
We have recently described a potent, orally bioavailable LTA4H inhibitor (LTA4H-I) that selectively blocks the production of LTB4 (20). In the present study, we evaluated the effect of this LTA4H inhibitor on allergen-induced airway responses in a mast cell–dependent murine model. In addition, we explored the mechanism of action of this inhibitor by examining effects on T-cell and dendritic-cell (DC) migration. Some of these results have been previously reported in the form of an abstract (21).
Balb/c mice (female, 6–8 wk old) were purchased from Charles River Laboratories (Hollister, CA). The animal handling and study protocol was approved by the Institutional Animal Care and Use Committee. Mice were sensitized with seven intraperitoneal injections of ovalbumin (OVA) and challenged three times with intranasal OVA as previously described (22); the study protocol is described in detail in the online supplement. Animals were orally dosed with a 20% cyclodextrin, vehicle (control), or JNJ 26993135, a selective LTA4H-I (10, 25, and 50 mg/kg twice daily) during the challenge phase and 30 minutes before OVA challenge. JNJ 26993135 was formulated as previously described (20). Anti-mouse IL-13 monoclonal antibody, kindly provided by Dr. William Glass (Centocor Inc, Malvern, PA) was administered twice intravenously during the challenge phase (500 μg) (23).
Airway function was measured using a small animal ventilator (Scireq, Montreal, Canada) as previously described (24). The airway function protocol is described in detail in the supplemental data section.
After killing, BAL samples were obtained as previously described (25). Cell-free supernatants were immediately frozen for subsequent cytokine analysis. Leukotriene C4 (LTC4) (Caymen 5205201), lipoxin A4 (LXA4) (Oxford Biomedical EA45), prostaglandin D2 (PGD2) (Caymen 512021), and LTB4 (Assay Designs 901-068) levels were measured from mouse BAL samples, as previously described (26).
Peribronchiolar lymph node (PBLN) cells and cells from phosphate-buffered saline (PBS)-perfused lungs were isolated and stimulated in quadruplicate as previously described (25, 27). Cytokine levels in cell culture supernatants were determined using Luminex multiplex system (Linco Research, Auburn, CA) as per manufacturer's protocol. Cytokine levels of interest were confirmed using individual ELISA assays (R&D, Minneapolis, MN).
Serum samples were obtained from animals by cardiac puncture. Total IgE, IgG1, and IgG2a levels were measured as per manufacturer's instructions (BD Biosciences, San Jose, CA) and are described in detail in the online supplement. To ensure assay linearity, serial dilutions of serum were assayed as described previously (25).
Mucin levels were detected by enzyme-linked lectin assay as previously described (28) using Ulex europaeus agglutinin-1 (UEA-1; Sigma, St. Louis, MO) and is described in detail in the online supplement.
Bone marrow-derived mast cell (BMMC) preparation and antitrinitrophenyl assay conditions are described in detail in the online supplement.
Dendritic cell migration was assayed as previously described (27) and is described in detail in the online supplement.
A one-way analysis of variance with a Bonferroni posttest or a Student t test was used to judge statistical significance as appropriate. In all cases the P values were calculated based on a comparison with vehicle-treated animals. The error bars shown represent the SD. In all cases the experiments were repeated two to three times with similar results and representative data are shown.
Using a recently described LTA4H inhibitor (20), we began by examining the efficacy of this compound and zileuton on primary, sensitized mast cells in response to IgE cross-linking ex vivo. We observed potent inhibition of LTB4 by both the LTA4H-I and zileuton (calculated inhibitory concentration of 50% of 34 nM and 354 nM, respectively, Figure 1A). Zileuton inhibited both LTC4 (IC50 of 209 nM) and LXA4 production (IC50 of 260 nM), whereas the LTA4H-I did not (Figures 1B and 1C). In fact, we observed a trend toward increased LXA4 production when BMMCs were treated with the LTA4H-I. We did not observe inhibition of PGD2 production (Figure 1C) or mast cell degranulation as assessed by β-hexosaminidase release (data not shown).
Figure 1. Effect of a leukotriene A4 hydrolase inhibitor (LTA4H-I) on murine bone marrow–derived mast cells (BMMCs). BMMCs were sensitized with antitrinitrophenyl IgE, preincubated with the indicated concentrations of an LTA4H-I and zileuton, and then stimulated with TNP. (A) Leukotriene B4 (LTB4), (B) leukotriene C4 (LTC4), (C) lipoxin A4 (LXA4), and (D) prostaglanin D2 (PGD2) levels in culture supernatant were measured by enzyme immunoassay. The vehicle controls for each assay (mean ± SD of four replicates) were 2,911 ± 77 pg/ml (LTB4), 15,433 ± 976 pg/ml (LTC4), 480 ± 23 pg/ml (LXA4), and 582 ± 40 ng/ml (PGD2).
[More] [Minimize]To study the role of LTA4H in an allergic lung inflammatory response, Balb/C mice were subjected to an OVA-induced lung inflammation protocol (26). Based on reports that BLT1-deficient mice have decreased levels of IL-13 in the BAL fluid (13, 14), we included an anti-mouse IL-13 antibody as well as the glucocorticoid steroid dexamethasone (dex) as a positive control in our studies. We used an invasive technique to examine lung function and AHR in a chronic, mast cell–dependent model (22). After methacholine provocation, OVA-challenged mice exhibited robust increases in all of the measured parameters compared with PBS-challenged animals (Table 1). As previously described (29), anti–IL-13 significantly decreased airway resistance (Figure 2A). Treatment with an LTA4H-I was statistically as efficacious as anti–IL-13 (Figure 2A) or dex, and exhibited a dose-dependent effect (Table 1).
Figure 2. (A–C) Methacholine dose–response relationships for airway resistance (R), tissue damping (G), and tissue stiffness (H). Each plotted value reflects the mean values for each group of mice (n = 6–10/group). Each animal's value reflects the mean of 12 sets of data captured over a 3-minute span after an aerosol methacholine challenge. Leukotriene A4 hydrolase inhibitor (LTA4H-I)-treated mice received a 50-mg/kg (mpk) dose, dexamethasone (dex)-treated mice received a 1-mpk dose, and anti–IL-13–treated mice received two 500-μg intravenous doses. Statistical significance of each treatment group compared with control vehicle-treated animals was calculated using a one-way analysis of variance (*P < 0.05; ***P < 0.001).
[More] [Minimize]R (cm H2O · s/ml) | C (cm H2O · s/ml) | RN (cm H20 · s/ml) | G (cm H2O/ml) | H (cm H2O/ml) | |
|---|---|---|---|---|---|
| Naive | 1.1 ± 0.2 | 0.0329 ± 0.0001 | 0.57 ± 0.15 | 5.97 ± 0.7 | 25 ± 2.2 |
| Vehicle | 10.5 ± 1.3 | 0.0066 ± 0.001 | 2.04 ± 0.41 | 92.3 ± 17 | 213 ± 26 |
| 10 mpk | 9.39 ± 1.2 | 0.0051 ± 0.0009 | 1.71 ± 0.19 | 82.1 ± 28 | 199 ± 37 |
| 25 mpk | 6.60 ± 1.2* | 0.0084 ± 0.003 | 1.53 ± 0.22* | 44.5 ± 17† | 68.0 ± 26† |
| 50 mpk | 2.79 ± 0.8† | 0.0131 ± 0.004* | 0.70 ± 0.07‡ | 17.6 ± 5.5‡ | 69.0 ± 18† |
| Anti–IL-13 | 1.65 ± 0.3‡ | 0.0245 ± 0.003‡ | 0.84 ± 0.13‡ | 9.17 ± 1.5‡ | 29.8 ± 2.9‡ |
| Dex | 1.23 ± 0.7‡ | 0.033 ± 0.009‡ | 0.55 ± 0.3‡ | 7.2 ± 3.3‡ | 26.3 ± 8.5‡ |
To examine potential changes in the lung, we also gathered data derived from the constant phase model of respiratory mechanics. Tissue damping (G was also significantly decreased in mice treated with an LTA4H-I, dex, or anti–IL-13 (Figure 2B). Other constant phase model parameters were also significantly decreased in mice treated with IL-13, dex, or LTA4H-I (Table 1). These parameters included Newtonian resistance (RN), a measure of resistance in the central airways, and tissue elastance (H), a measure of tissue stiffness comprising both the parenchymal and peripheral airways (Figure 2C).
Inflammatory leukocyte influx into the lung lumen was confirmed by dramatically increased cell counts in BAL fluid from OVA-challenged versus naive mice (Figure 3). Treatment with an LTA4H-I significantly decreased total cells, neutrophils, and lymphocytes in the BAL fluid in a dose-dependent manner (Figures 3A–3D). Highly significant decreases in eosinophil numbers (P < 0.001) were also observed (Figure 3B). Treatment with anti–IL-13 antibody only modulated eosinophilia, whereas treatment with dex was generally antiinflammatory except for the neutrophilia. We observed no change in macrophage cell counts with either LTA4H-I or anti–IL-13 treatment (data not shown).
Figure 3. Dose–response relationship of a leukotriene A4 hydrolase inhibitor, anti–IL-13, and dexamethasone (dex) on airway inflammation in Balb/C mice (n = 8–10 animals/group). (A) The total number of cells and a differential cell count for (B) eosinophils, (C) neutrophils, and (D) lymphocytes were calculated from BAL fluid collected after the final ovalbumin challenge. (*P < 0.05; **P < 0.01; ***P < 0.001; one-way analysis of variance.) mpk = mg/kg.
[More] [Minimize]We next examined relevant chemokine and Th2 cytokine levels in the BAL samples. The LTA4H-I dose-dependently decreased the levels of a panel of inflammatory cytokines and chemokines (Table 2). Significant reductions in IL-5, eotaxin, and monocyte chemoattractant protein-1 were observed, with highly significant reductions seen in IL-4 and IL-13 (Figure 4A). We did not observe a change in IFN-γ levels with inhibitor treatment, suggesting that there was no general skewing of the immune system to a Th1 response. Given the dramatic decrease observed in BAL IL-13 levels on treatment with an LTA4H-I, we next focused on T-cell production of IL-13 in response to ex vivo stimulation with OVA. Lung T cells produced high levels of IL-13 on restimulation, and LTA4H-I treatment in vivo resulted in a significant decrease in IL-13 production ex vivo (Figure 4B). In the case of T cells from PBLN, both nonstimulated and OVA-stimulated T cells produced significantly decreased levels of IL-13 (Figure 4C). We observed no proliferative defect of these T cells to antigen ex vivo (data not shown).
Figure 4. (A) A leukotriene A4 hydrolase inhibitor (LTA4H-I) decreases Th2-associated cytokines and chemokines in bronchoalveolar lavage (BAL) fluid. Cell-free BAL fluid from vehicle and inhibitor-treated mice (50-mg/kg [mpk] dose group) was assayed for the indicated cytokines (n = 8–10 animals/group). Ex vivo restimulation of T cells isolated from (B) lung or (C) peribronchiolar lymph nodes of vehicle or LTA4H-I–treated mice. Cells were isolated and cultured without stimulation (−) or stimulated with ovalbumin (OVA) (+) as described in the Methods. (D) Dose–response relationship of an LTA4H inhibitor and anti–IL-13 on mucin levels in the BAL fluid. Mucin levels were quantified using an enzyme-linked lectin assay as described in the methods. (*P < 0.05; **P < 0.01; ***P < 0.001; one-way analysis of variance.)
[More] [Minimize]IL-4 | IL-5 | IL-13 | IFN-γ | MCP-1 | Eotaxin | |
|---|---|---|---|---|---|---|
| Naive | 3 ± 4 | 6 ± 3 | 3 ± 2 | 2 ± 4 | 1 ± 1 | 0 |
| Vehicle | 95 ± 29 | 102 ± 42 | 538 ± 133 | 28 ± 11 | 130 ± 35 | 131 ± 54 |
| 10 mpk | 78 ± 23 | 80 ± 51 | 295 ± 95* | 27 ± 13 | 126 ± 52 | 126 ± 61 |
| 25 mpk | 59 ± 32* | 71 ± 36 | 153 ± 59† | 33 ± 16 | 82 ± 36* | 109 ± 50 |
| 50 mpk | 35 ± 18‡ | 51 ± 19* | 42 ± 53† | 31 ± 13 | 41 ± 20† | 63 ± 41* |
| Anti–IL-13 | 102 ± 39 | 68 ± 34 | 796 ± 298 | 36 ± 23 | 96 ± 44 | 80 ± 41 |
Next, we used a lectin-based assay to measure levels of mucin in the lung lumen contents. We observed a significant increase in mucus levels in the BAL fluid of vehicle-treated, OVA-challenged animals when compared with naive control animals (Figure 4D). Treatment with an anti–IL-13 antibody was extremely efficacious in decreasing mucin levels. Mucin levels were also significantly decreased in mice treated with an LTA4H-I in a dose-dependent manner (Figure 4D).
When compared with a traditional OVA model, the mast cell–dependent model results in much higher levels of total serum IgE (115 ng/ml vs. 665 ng/ml) and OVA-specific IgE (40 ng/ml vs. 311 ng/ml) (data not shown). Although extremely effective at decreasing AHR, an anti–IL-13 antibody did not significantly decrease IgE and IgG1 levels (Figure 5). In contrast, treatment with an LTA4H-I resulted in a statistically significant, dose-dependent decrease in total serum IgE as well as OVA-specific IgG1 and IgE (Figures 5A–5C). We did not observe a marked increase in IgG2A in this model (Figure 4D). Collectively, these data indicated that an LTA4H-I affected multiple features of allergic airway inflammation.
Figure 5. Dose–response relationship of a leukotriene A4 hydrolase (LTA4H) inhibitor and anti–IL-13 on antibody levels in serum from each treatment group (n = 6–10 mice). Serum samples were collected by cardiac puncture and assayed by ELISA for the presence of (A) total IgE, (B) ovalbumin (OVA)-specific IgE, (C) OVA-specific IgG1, and (D) OVA-specific IgG2A. (*P < 0.05; **P < 0.01; ***P < 0.001; one-way analysis of variance.) mpk = mg/kg.
[More] [Minimize]Based on the findings above, we decided to explore the mechanism by which an LTA4H inhibitor might affect leukocyte trafficking in the allergic airways. Recent studies using BLT1-deficient mice have indicated that LTB4 plays a key role in orchestrating lung inflammation by regulating effector T-cell recruitment (13, 30). Treatment of mice with an LTA4H-I significantly reduced the numbers of both CD4+ and CD8+ T cells in the lung tissue (Figures 6A and 6B) and lumen (Figure 3D). However, we did not observe a change in the number of CD4+ and CD8+ T cells in PBLN or spleen (Figures 6C and 6D and data not shown).
Figure 6. Dose–response relationship of a leukotriene A4 hydrolase (LTA4H) inhibitor on T cells in the lung and draining peribronchiolar lymph nodes. T cells were purified from lung and lymph nodes as described in methods (n = 6–8 animals/group). The number of T cells was measured using CD3, CD4, and CD8 surface staining by fluorescence-activated cell sorter. (*P < 0.05; **P < 0.01; ***P < 0.001; one-way analysis of variance.) mpk = mg per kg; PBLN = peribronchiolar lymph node.
[More] [Minimize]Although we did not observe a decrease in total T cells in PBLN, ex vivo restimulation of T cells from either lung or PBLN resulted in decreased cytokine production, such as IL-13 (Figures 4B and 4C). These observations raised the possibility that blocking LTB4 in the lung might regulate T-cell priming in the draining lymph node (31). To directly test whether inhibition of LTA4H effects DC migration, we challenged OVA-sensitized mice with Alexa488-OVA via intranasal instillation and monitored the trafficking of Alexa488+ DC subsets to the lung-draining lymph nodes and the lung (Figure 7). Treatment with an LTA4H-I decreased migration of CD8α− DCs to the PBLNs and the lung (Figures 7B and 7C). As has been reported for BLT1-deficient DCs in vitro (32), blocking LTB4 in vivo resulted in a decrease of CCR7+ DCs in the draining lymph nodes and the lung (Figures 7B and 7C).
Figure 7. Inhibition of leukotriene B4 (LTB4) production decreases lung dendritic cell (DC) migration to draining lymph nodes. Mice were challenged three times with ovalbumin (OVA), the third time being with Alexa488-OVA. Lungs and draining lymph node cells were collected 18 hours later. Two groups (n = 6–8/group) consisting of vehicle or leukotriene A4 hydrolase inhibitor (LTA4H-I)-treated mice (50-mg/kg dose) were analyzed. (A) Dead cells were excluded based on PI staining and Alexa488+ cells were gated for further analysis. (B). Total numbers of Alexa488+ DC subsets from (B) the lymph node and (C) the lung expressing CD8α or CCR7 were calculated based on the percentage of positive cells and total number of cells analyzed. FSC = forward scatter, PI = propidium iodide, SSC = side scatter.
[More] [Minimize]After challenge with antigen, BAL fluid from vehicle-treated mice contained detectable levels of LTB4 (232 ± 26 pg/ml, mean ± SD) (Figure 8A). Treatment with an LTA4H-I dose-dependently decreased the levels of LTB4 detectable in cell-free BAL fluid, whereas BAL fluid from PBS-challenged mice had undetectable levels of LTB4. Another potential mechanism by which LTA4H-I might regulate AHR and lung inflammation involves increased production of lipoxins. We detected a dose-dependent significant increase in LXA4 levels in the BAL fluid of mice treated with an LTA4H-I. As expected, use of the 5-LO inhibitor zileuton blocked LTB4 production but did not increase LXA4 levels (Figure 8). As a control, we measured levels of LTC4 and observed a significant decrease in zileuton-treated mice and no modulation with an LTA4H-I (Figure 7C).
Figure 8. Inhibition of leukotriene A4 (LTA4) hydrolase decreases leukotrienes and increases lipoxins in the bronchoalveolar lavage (BAL) fluid. Mice were challenged with ovalbumin (OVA) and BAL fluid was collected 24 hours later. Cell-free BAL fluid from mice treated with vehicle, leukotriene A4 hydrolase inhibitor (10, 25, and 50 mg/kg [mpk]), and zileuton (50 mpk) (n = 8 mice/group) were assayed for (A) leukotriene B4 (LTB4) levels, (B) lipoxin A4 (LXA4) levels, and (C) leukotriene C4 (LTC4) levels. (*P < 0.05; **P < 0.01; ***P < 0.001; one-way analysis of variance.)
[More] [Minimize]Inflammatory leukocytes, including mast cells, macrophages, and neutrophils, can produce LTB4, a potent chemoattractant for multiple leukocytes. Elevated levels of LTB4 have been reported in a number of inflammatory airway disorders, including sputum and BAL samples from patients with chronic obstructive pulmonary disease, allergy, and asthma (8–10).
Because of conflicting efficacy results obtained from acute lung inflammation studies using BLT1-deficient mice (13, 14), we chose to use a selective LTA4H inhibitor to pharmacologically evaluate the contribution of LTB4 to allergic lung inflammation. The LTA4H-I, JNJ 26993135, was able to potently inhibit LTB4 production by murine BMMCs triggered via the IgE receptor. Moreover, using this pharmacological inhibitor of LTB4 production around the challenge phase of a mast cell–dependent OVA model to mimic a therapeutic situation, we observed dramatic decreases in airway inflammation and a reduction in markers of airway remodeling and function.
Previous work has demonstrated that individual airway function parameters obtained using the forced oscillation technique can be used to measure specific changes in lung mechanics and pathology (33, 34). Herein, treatment with an LTA4H inhibitor decreased all three parameters generated using the constant-phase model in response to methacholine challenge, central airways Newtonian resistance (RN), peripheral tissue damping (G), and tissue rigidity (H). The highly significant effects on G suggest changes in the resistance of lung parenchyma or very small airways are decreased on inhibition of LTB4 production. Likewise, effects on H suggest inhibition of LTB4 production results in decreased airway closure. Together, these effects on both G and H suggest that LTB4 inhibition results in decreased dysfunction of the peripheral airways. This hypothesis involving enhanced lung function due to reduced airway closure is further supported by our findings that inhibition of LTB4 production results in dramatic reductions in Th2 cytokines, such as IL-13 and IL-4. Decreases in these cytokines would result in decreased goblet cell hyperplasia and the resulting production of mucin (35). Indeed, we observed significantly decreased levels of mucin in BAL fluid of mice treated with an LTA4H-I or anti–IL-13. This mechanism of airway closure due to mucus hypersecretion is also reflected by clinical observations in the population with severe asthma (36).
A model has recently been proposed to explain how LTB4 controls AHR (37); while this manuscript was in preparation, a role for LTA4H in mast cell LTB4 formation and allergic airway inflammation was reported using LTA4H knockout mice (38). Antigen-triggered activation of mast cells via the IgE receptor results in localized production of LTB4 in the lung. LTB4 then recruits antigen-specific BLT1+ T cells that produce cytokines such as IL-4 and IL-13 (13, 30). Interestingly, our studies using a more chronic model of airway inflammation demonstrated that inhibition of LTB4 production during the challenge phase affects multiple parameters of this model. We observed a dramatic decrease in antigen-specific IgE and IgG1 that would ultimately modulate the activation of mucosal mast cells and thus may further decrease the localized production of LTB4 in the lung on subsequent antigen challenges.
Tager and colleagues (4) and Goodarzi and colleagues (39) demonstrated that effector T-cell recruitment into the inflamed lung is driven by LTB4. Data using BLT1-deficient animals and adoptive transfer studies using BLT1-deficient T cells have indicated that both CD4+ and CD8+ effector T cells play a key role in inflammation and AHR (13, 30). In agreement with these observations, an LTA4H-I in our studies significantly reduced the numbers of both CD4+ and CD8+ T-cell subsets in the lung tissue and lumen with a concomitant reduction in airway IL-4, IL-13, and AHR. Based on these findings, we hypothesize that blocking LTB4 production with a pharmacological inhibitor results in decreased accumulation of both CD4+ and CD8+ T cells in the lung tissue and lumen, through a direct effect on mast cell production of LTB4.
Despite similar numbers of T cells in PBLN, we did observe a dramatic decrease in cytokine production when PBLN T cells were restimulated ex vivo. This prompted us to examine the potential effects of LTB4 on DCs. Lung DCs have been shown to play a key role in driving lung inflammation and AHR on allergen challenge (31). In addition, studies using DCs from BLT1-deficient mice have suggested that LTB4 plays a role in DC migration to draining lymph nodes in a contact hypersensitivity model (32). We gated on CD11c+ DCs, a marker expressed by both airway and parenchymal DCs that are known to be more endocytic and efficient at presenting peptides to T cells (40). The use of an LTA4H-I during the challenge phase effectively decreased the migration of CD11c+, Alexa488+ DCs into the PBLN. Decreased migration was observed with several subpopulations of lung DCs, including CD8α− and CCR7+ DCs. These are the first data, to our knowledge, that demonstrate a role for LTB4-induced CDα− DC migration in the allergic lung. CD8α− DCs have been demonstrated to be the dominant OVA-processing and presenting DC population in PBLNs after challenge with OVA (27). It has been previously demonstrated that CCR7 is an important chemokine receptor for pulmonary DC trafficking to draining lymph nodes (41), and its expression on DCs can be modulated by LTB4 (32). These data suggest that blocking LTB4 production in the lung could possibly decrease CD4+ and CD8+ T-cell activation in draining lymph nodes by modulating the migration of DCs that have taken up allergen in the lung. It remains to be determined whether this modulation results in a decrease in the precursor frequency of antigen-specific effector T cells or a defect in the ability of these T cells to produce Th2 cytokines.
Another potential mechanism by which LTA4H-I might regulate AHR and lung inflammation involves increased production of lipoxins. We detected significantly increased LXA4 levels in the BAL fluid of mice treated with an LTA4H-I, a phenomenon that may result from shunting of LTA4H substrates to the lipoxin pathway (20). LXA4 and its analogs have been shown to compete for binding of LTD4 with CysLT1 (42). In addition, LXA4 has the potential to directly affect airway smooth muscle on antigen challenge, both in vitro to LTD4-induced constriction in animal models of AHR and in patients with asthma challenged with LTC4 (43–45). Conceivably, the observed increases in LXA4 could therefore account for some of the results reported herein and enhance the therapeutic benefits of LTA4H-I in the clinic. Previous studies with this LTA4H-I have importantly demonstrated no such shunting to inflammatory LTC4 production in ionophore-stimulated whole blood or in an acute peritonitis model in mice (20). Likewise, as one would expect from a selective LTA4H-I, no inhibition of LTC4 was observed here in the lung or previously in the peritoneum (20). In contrast, the 5-LO inhibitor zileuton was effective at inhibiting both LTB4 and LTC4 in the BAL fluid of OVA-challenged animals, although the effect on LTC4 was less dramatic. This pharmacodynamic effect has been reported clinically; in allergen-challenged patients, zileuton was able to inhibit LTB4 in ionophore-stimulated whole blood, but only partially blocked LTE4 in the urine, a predictive marker of LTC4 synthesis (46).
A recent study with zileuton has reaffirmed its ability to significantly improve asthma symptoms and reduce exacerbations and concomitant drug usage (47). Whether this activity, or purported improved efficacy over CysLT1 antagonists in more severe asthma, is related to its inhibition of LTB4 formation remains to be elucidated, but the preferential effect on LTB4 production is provocative. Novel inhibitors of LTA4H, dosed alone or in conjunction with CysLT1 antagonists, may help define the specific role of LTB4 in asthma.
In summary, a novel and selective LTA4H inhibitor can modulate airway inflammation and AHR through multiple mechanisms. Consistent with direct inhibitory effects of LTB4, T-cell and DC chemotaxis were inhibited. Correspondingly, reductions in Th2 cytokine–driven pathologies, such as mucus secretion and AHR, were also attenuated. Additional, novel benefits of LTA4H inhibition were also observed, including an increase in antiinflammatory lipoxin levels in the lung. These data suggest that LTA4H inhibition might be useful in treating allergic airway diseases.
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