All the mice were bred and housed in a specific pathogen-free barrier facility maintained by the University of Chicago Animal Resources Center. The studies reported here conform to the principles outlined by the Animal Welfare Act and the National Institutes of Health guidelines for the care and use of animals in biomedical research.

Extract-N-Amp Tissue PCR kit (Sigma Aldrich, S. Louis) was utilized for isolating genomic DNA from mouse tail and amplifying DNA fragments. The primers for LPA₁ and LPA₂ knockout mice were described as previous studies 3334.

Schistosoma mansoni eggs sensitization and challenge to induce murine allergic airway disease were described before 31. In brief, at day 0, mice (6-8 weeks) were immunized by i.p. injection of 5,000 inactivated Schistosoma mansoni eggs. At day 7, the mice were challenged with 10 μg of SEA by intratracheal aspiration. The mice were studied at day 11.

BAL fluids were performed by an intratracheal injection of 1 ml of PBS solution followed by gentle aspiration. The lavage was repeated twice to recover a total volume of 1.8-2.0 ml. The lavage was centrifuged and supernatant was processed for PGE2 or LPA measurement. The percentages of cell types in BAL fluids were determined by FACS analysis with cell type-specific markers.

Lungs were removed from mice and lobes were sectioned sagitally, embedded in paraffin, cut into 5-μm sections. Periodic Acid Schiff (PAS) staining were performed by Pathology Core Facility in The University of Chicago.

Antibody to mouse CCR3 (clone 831101.111) was obtained from R&D Systems (Minneapolis, MN). Cells were fixed with 4% paraformaldehyde for 10 min and incubated with staining antibodies for 30 min at 4°C. The samples were washed and analyzed on a FACS LSR-II (Becton Dickinson).

Briefly, mice were euthanized and their tracheas were isolated and digested with 0.1% protease (Type XIV, Sigma) overnight at 4°C. The tracheal cell suspension were transferred to 15 ml tube and spun at 1500 rpm for 3 min at 4°C and were pooled in BEGM medium (Lonza, Walkersville, MD).

Lipids in BAL were extracted as described before 28. In brief, LPA levels were determined using liquid chromatography and tandem mass spectrometry (LC) with ABI-4000 Q-TRAP hybrid triple quadrupole/ion trap mass spectrometer (MS) coupled with an Agilent 1100 liquid chromatography system. Lipids were separated using methanol/water/HCOOH, 79/20/0.5, v/v, with 5 mM NH4COOH as solvent A and methanol/acetonitrile/HCOOH, 59/40/0.5, v/v, with 5 mM NH4COOH as Solvent B. LPA molecular species were analyzed in negative ionization mode with declustering potential and collision energy optimized for LPA.

Mouse tracheal epithelial cells grown on 6-well plates were challenged with LPA for 3 h, medium were collected and centrifuged at 5,000 × g for 10 min at 4°C. The supernatant or BAL fluid supernatant were transferred to new 2.0 ml-eppendorf tubes and frozen in -80°C for later analysis. Measurement of PGE2 levels, as 13, 14-dihydro-15-keto PGE2, was carried out using a commercial ELISA kit according to manufacture's instruction.

Total RNA was isolated from cultured mouse tracheal epithelial cells using TRIzol^® reagent (Life Technology, Rockville, MD) according to the manufacturer's instructions. RNA was quantified spectrophotometrically and 1 μg of RNA was reversed transcripted using cDNA synthesis kit (Bio-Rad) and Real-time PCR and quantitative PCR were performed to assess expression of the COX-2, LPA₁, LPA₂, LPA₃, LPA₄, and LPA₅ using primers designed based on mouse mRNA sequences (Table 1.). Amplicon expression in each sample was normalized to its 18S RNA content. The relative abundance of target mRNA in each sample was calculated as 2 raised to the negative of its threshold cycle value times 10⁶ after being normalized to the abundance of its corresponding 18S, [e.g., 2 ^{-(Target Gene Threshold Cycle)}/2 ^{-(18S Threshold Cycle)} × 10⁶].

Equal amounts of protein (20 μg) were subjected to 10% SDS/PAGE gels, transferred to polyvinylidene difluoride membranes, blocked with 5% (w/v) BSA in TBST (25 mM Tris-HCl, pH 7.4, 137 mM NaCl and 0.1% Tween-20) for 1 h and incubated with anti-COX-2 antibody in 5% (w/v) BSA in TBST for 1-2 h at room temperature. The membranes were washed at least three times with TBST at 15 min intervals and then incubated with a rabbit horseradish peroxidase-conjugated secondary antibody (1: 3,000) for 1 h at room temperature. The membrane was developed with enhanced chemiluminescence detection system according to Manufacturer's instructions.

All results were subjected to statistical analysis using one-way ANOVA and, whenever appropriate, analyzed by Student-Newman-Keuls test. Data are expressed as means ± S.D. of triplicate samples from at least three independent experiments and level of significance was taken to P < 0.05.

To investigate the role of LPA receptors in pathogenesis of asthma, we quantified LPA levels in BAL fluids from control and SEA-challenged mice. Mice were sensitized by i.p. injection of 5,000 inactivated Schistosoma mansoni eggs. At day 7, mice were challenged with or without 10 μg of SEA by intratracheal aspiration and at day 11, BAL fluids were collected (Fig. 1) and lipid were extracted and LPA levels in BAL fluids were measured by LC-MS/MS with C17:0 LPA as an internal standard. As shown in Table 2, LPA was detectable (~1254.3 ± 357.0 pmole/ml) in control mice (sensitized with inactivated Schistosoma mansoni eggs but not SEA challenged), and there was a ~2.8 fold increase in LPA levels (~3557.9 ± 109.3 pmole/ml) in Schistosoma mansoni eggs sensitized and challenged mice, compared to control mice. Unsaturated molecular species of LPA (18:1, 20:4, 22:5, and 22:6) were detected in BAL fluids of control mice, which increased significantly after SEA challenge. These results show for the first time, to our knowledge, increase in LPA during allergic lung inflammation in a murine model of asthma.

To determine the role of LPA receptors in airway inflammation mediated by Schistosoma mansoni eggs sensitization and challenge, we used LPA₁ and LPA₂ deficient mice, which were genetically engineered as described earlier 3334. The heterozygous LPA₁^+/- and LPA₂^+/- mice were housed and bred at the University of Chicago Animal Resources Center and described experiments were approved by the ACIU of the University of Chicago. Genotyping analyses with specific primers confirmed generation of wild type (+/+), heterozygous (+/-) and homozygous mice (-/-) from the genetically engineered LPA₁ and LPA₂ mice (data not shown). Since LPA₁^-/- showed 50% neonatal lethality and impaired sucking in neonatal pups, all experiments were carried out with LPA₁^+/- and LPA₂^+/- mice to investigate the role of LPA receptors in Schistosoma mansoni eggs sensitization and challenge-mediated allergic inflammatory responses. To determine whether LPA₁^+/- and LPA₂^+/- mice reduced the effect of LPA, wild type, LPA₁^+/- and LPA₂^+/- mice were intratracheal challenged with 18:1LPA (5 μM in 25 μl PBS) for 6 h. As shown in Fig. 2, LPA challenge increased neutrophil infiltration, however, LPA₁^+/- and LPA₂^+/- mice reduced LPA-induced neutrophil infiltration in BAL fluids, suggesting that less LPA₁ and LPA₂ receptors in LPA₁^+/- and LPA₂^+/- mice reduce LPA-induced inflammation in lung and that LPA₁^+/- and LPA₂^+/- mice are useful models for investigating role of LPA receptors in lung inflammatory diseases.

Wild type, LPA₁^+/-, and LPA₂^+/- mice were sensitized with inactivated Schistosoma mansoni eggs and challenged with or without SEA for 4 days, BAL fluids and lung tissues were collected, cell numbers were measured under microscope and total eosinophils were determined by flow cytometry using eosinophils specific antibody (anti-CCR3). Consistent with pervious reports 3032, Schistosoma mansoni eggs sensitized and challenged wild type mice showed significant increase in total cell numbers and eosinophils in BAL fluids; however, total cell numbers and recruitment of eosinophils were attenuated in LPA₂^+/-, but not LPA₁^+/- mice (Fig. 3). These results suggest a role for LPA₂ in influx of eosinophils into alveolar space during allergic inflammatory response.

Airway goblet cell metaplasia and mucus production, indices of degree of inflammation, are hallmarks of asthma. Goblet cell metaplasia and mucus production were determined by PAS staining of histological sections of lung tissues from Schistosoma mansoni eggs sensitized and challenged or non-challenged wild type, LPA₁^+/-, and LPA₂^+/- mice. As shown in Fig. 4A, PAS positive goblet cells were higher in Schistosoma mansoni eggs sensitized and challenged wild type mice, compared to Schistosoma mansoni eggs sensitized and non-challenged wild type mice (control mice), whereas significantly less PAS stained goblet cells were seen in Schistosoma mansoni eggs sensitized and challenged LPA₁^+/- and LPA₂^+/- mice, compared to Schistosoma mansoni eggs sensitized and challenged wild type mice. Scoring of the histological sections also confirmed a significantly higher percentage of bronchi for PAS positive stained cells in the sensitized and challenged control wild type mice compared to LPA₁^+/- and LPA₂^+/- mice (Fig. 4B). These results demonstrate that Schistosoma mansoni eggs sensitized and challenged LPA₁^+/- and LPA₂^+/- mice develop reduced goblet cell metaplasia and mucus production compared to control wild type mice. Together, these data suggest a role for LPA receptors for optimal induction of Th2-mediated airway inflammation.

Endogenous PGE2 is produced by airway epithelium, smooth muscle, dendritic cells, and macrophages in response to allergen challenge 35. PGE2 has been shown to be an anti-inflammatory lipid mediator and bronchodilator in the airway 2425; however, administration of PGE2 induced various side effects, including cough, enhanced mucus production, and sensory nerve stimulation 36. To determine the role of LPA receptors expression and PGE2 production in response to allergen challenge, we analyzed PGE2 levels in BAL fluids and COX-2 expression in lung tissues from Schistosoma mansoni eggs sensitized and challenged wild type mice. As shown in Fig. 5A, PGE2 levels were higher in control wild type and LPA₁^+/- mice, compared to LPA₂^+/- mice in response to Schistosoma mansoni eggs sensitization and challenge. Schistosoma mansoni eggs sensitization and challenge increased COX-2 expression in lung tissues of wild type mice while LPA₂^+/- mice showed reduced COX-2 expression (Fig. 5B). Recently, we have shown that LPA induces COX-2 expression and PGE2 release in human bronchial epithelial cells 23. As Schistosoma mansoni eggs sensitization and challenge increased LPA levels in BAL fluids (Table 2), we measured LPA levels in BAL fluids from sensitized and SEA challenged LPA₂^+/- mice. Compared to wild type mice, LPA levels in BAL fluids from LPA₂^+/- mice were decreased after SEA challenge. Together, these results suggest that increased lung COX-2 expression, PGE2 and LPA production in BAL fluids by Schistosoma mansoni eggs sensitization and challenge is regulated by LPA₂.

Having demonstrated a role for LPA₂ in Schistosoma. mansoni eggs-induced COX-2 expression, PGE2 secretion and airway inflammation, we hypothesized that expression of LPA₂ on airway epithelial cells may be involved in inflammatory responses to Schistosoma. mansoni eggs sensitization and challenge. To investigate the role of LPA₂ in LPA-induced COX-2 expression and PGE2 production, tracheal epithelial cells were isolated from wild type and LPA₂^+/- mice. Analysis of total RNA for mRNA expression of LPA receptors by real-time RT-PCR revealed that expression of LPA₂>LPA₄>LPA₁ ≥ LPA₃ in mouse tracheal epithelial cells (Table 3). In contrast to mouse tracheal epithelial cells, LPA₁ and LPA₃ were predominantly expressed in human bronchial epithelial cells 37. In LPA₂^+/- tracheal epithelial cells, expression of LPA₂ mRNA was reduced to ~50%, compared to wild type mice, while there were no significant changes in expression levels of LPA₁ and LPA₃ mRNA (Fig. 6A). To determine the role of LPA₂in LPA mediated COX-2 expression and PGE2 release, tracheal epithelial cells from wild type and LPA₂^+/- mice were challenged with LPA (1 μM) for 3 h, total RNA isolated and COX-2 mRNA expression determined by Real-time RT-PCR. LPA stimulated COX-2 mRNA expression in wild type mouse cells (~13 fold); however, LPA-induced COX-2 mRNA expression was reduced in LPA₂^+/- mouse cells (~56% of wild type cells) (Fig. 6B). The media, after LPA challenge, were collected and PGE2 levels were determined. As shown in Fig. 6C, PGE2 release from LPA₂^+/-mouse tracheal epithelial cells challenged with LPA was lower as compared to cells from wild type mice [PGE2 (pg/ml)-Wild type: vehicle, 268 ± 29; LPA, 432 ± 47; LPA₂^+/-: vehicle, 283 ± 21; LPA, 374 ± 16]. These results suggest that a role for LPA₂ in LPA-induced COX-2 expression and PGE2 release from mouse tracheal epithelial cells.