Lysophosphatidic acid receptor 1 modulates lipopolysaccharide-induced inflammation in alveolar epithelial cells and murine lungs

Abstract

Lysophosphatidic acid (LPA), a bioactive phospholipid, plays an important role in lung inflammation by inducing the release of chemokines and lipid mediators. Our previous studies have shown that LPA induces the secretion of interleukin-8 and prostaglandin E(2) in lung epithelial cells. Here, we demonstrate that LPA receptors contribute to lipopolysaccharide (LPS)-induced inflammation. Pretreatment with LPA receptor antagonist Ki16425 or downregulation of LPA receptor 1 (LPA(1)) by small-interfering RNA (siRNA) attenuated LPS-induced phosphorylation of p38 MAPK, I-κB kinase, and I-κB in MLE12 epithelial cells. In addition, the blocking of LPA(1) also suppressed LPS-induced IL-6 production. Furthermore, LPS treatment promoted interaction between LPA(1) and CD14, a LPS coreceptor, in a time- and dose-dependent manner. Disruption of lipid rafts attenuated the interaction between LPA(1) and CD14. Mice challenged with LPS increased plasma LPA levels and enhanced expression of LPA receptors in lung tissues. To further investigate the role of LPA receptors in LPS-induced inflammation, wild-type, or LPA(1)-deficient mice, or wild-type mice pretreated with Ki16425 were intratracheally challenged with LPS for 24 h. Knock down or inhibition of LPA(1) decreased LPS-induced IL-6 release in bronchoalveolar lavage (BAL) fluids and infiltration of cells into alveolar space compared with wild-type mice. However, no significant differences in total protein concentration in BAL fluids were observed. These results showed that knock down or inhibition of LPA(1) offered significant protection against LPS-induced lung inflammation but not against pulmonary leak as observed in the murine model for lung injury.

Fig. 1.

Inhibition or downregulation of LPA₁ attenuates LPS-induced signaling. A: MLE12 cells were treated with Ki16425 (1, 5, and 10 μM, 1 h) before LPS challenge (10 μg/ml, 1 h). Equivalent amounts of cell lysates were subjected to immunoblotting with antibodies against phosphorylated (p)-p38, p-38, p-I-κB kinase (IKK), IKK, p-IκB, and β-actin. Representative blots from 3 independent experiments are shown. DMSO, dimethyl sulfoxide. B: relative expression levels of the above proteins as evidenced from density profiles using image J software. C: MLE12 cells were transfected with control small-interfering RNA [siRNA (siCont)] or LPA₁ siRNA (siLPA₁, 50 ng/ml, 72 h) before LPS challenge (10 μg/ml) for 1 h. Cell lysates were analyzed by immunoblotting using antibodies against p-p38, p-38, p-IKK, IKK, p-IκB, and β-actin. Representative blots from 3 independent experiments are shown. D: intensity changes of phosphoproteins in the immunoblots shown in C were analyzed by image J software. Veh, vehicle.

Fig. 2.

Inhibition or downregulation of LPA₁ attenuates LPS-induced IL-6 secretion. A: MLE12 cells were pretreated with Ki16425 (1–10 μM, 1 h) before LPS challenge (10 μg/ml, 3 h). IL-6 levels in culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA). B: MLE12 cells were transfected with siCont or siLPA₁ (50 ng/ml, 72 h) before LPS challenge (10 μg/ml) for 3 h, and IL-6 levels in culture supernatants were measured by ELISA. Inset shows the effect of siLPA₁ on expression of LPA₁ based on immunoblotting whole cell lysates using anti-LPA₁ antibody.

Fig. 3.

LPS-induced phosphorylation of p38 MAPK and I-κB is independent of lysophosphatidic acid (LPA) generation. A: MLE12 cells were treated with LPA (0.1–1 μM) for 3 h, and IL-6 levels in culture supernatants were measured by ELISA. B: MLE12 cells (∼60% confluence) were infected with adenoviral vector control or adenoviral mouse lipid phosphate phosphatase 1 (LPP-1) wild type at different multiplicities of infection (MOI) (0, 5, 25, or 50) for 48 h. The virus-containing medium was replaced with DEEM-F-12 medium, and cells were challenged with LPS (10 μg/ml) for 1 h. Equivalent amounts of cell lysates were subjected to Western blotting with antibodies against p-p38, p-38, p-IκB, β-actin, and Myc. Representative blots from 3 independent experiments are shown. C: relative expression levels of the above proteins as evidenced from density profiles using image J software. D: 1 μM of 18:1 LPA was added to cell cultures of MLE12 cells transfected with adenoviral vector control or adenoviral mLPP-1 wild type (50 MOI, 48 h) for 1 h. Supernatant was collected, and LPA levels in supernatant were measured by LC-MS/MS.

Fig. 4.

LPS increases LPA₁ interaction with CD14. A: MLE12 cells were incubated with neutralizing CD14 antibody (Ab, 10 μg/ml) or an equivalent amount of IgG for 6 h before LPS (10 μg/ml, 1 h) challenge. Equivalent amounts of cell lysates were subjected to immunoblotting with antibodies against p-p38, p-38, p-IκB, and β-actin. Representative blots from 3 independent experiments are shown. B: relative expression levels of the above proteins as evidenced from density profiles using image J software. C: MLE12 cells treated with LPS (1, 10, or 50 μg/ml) for 2 h; CD14-containing protein complexes were immunoprecipitated (IP) using a CD14-specific antibody and analyzed by immunoblotting (IB) with antibodies to LPA₁ and CD14. Representative blots from 3 independent experiments are shown. D: MLE12 cells were treated with LPS (10 μg/ml) for 0.5, 1, and 2 h, CD14 immunoprecipitated with an anti-CD14 antibody, and analyzed for coimmunoprecipitated proteins. Representative blots from 3 independent experiments are shown. E: MLE12 were treated with 5 mM methyl-β-cyclodextrin (MBCD) for 2 h before LPS challenge (10 μg/ml) for 2 h. LPA₁ was immunoprecipitated with an anti-LPA₁ antibody and analyzed for coimmunoprecipitated proteins. Representative blots from 2 independent experiments are shown. F: MLE12 cells grown on a glass chamber were treated with LPS (10 μg/ml) for 2 h, fixed using 3.7% formaldehyde for 20 min, followed by a wash in PBS containing 0.1% Triton for 1 min. LPA₁ and CD14 were immunostained with antibodies to LPA₁ and CD14. Fluorescence intensity profiles are also shown. Green fluorescence is for LPA₁ signal, red fluorescence is for CD14 signal, and blue fluorescence is for nuclei signal. Arrows show colocalization of LPA₁ and CD14. Shown are representative data from 3 independent experiments.

Fig. 5.

LPS increases the expression LPA receptors in lung tissue. Mice were intratracheally challenged with LPS (5 mg/kg body wt) for 24 h. The lung tissue total RNA was extracted, and mRNA levels of LPA₁ (A), LPA₂ (B), and LPA₃ (C) were determined by real-time RT-PCR. LPA_1–3 protein expression were examined by immunoblotting (D). Relative expression levels of the above proteins as evidenced from density profiles using image J software.

Fig. 6.

LPA₁^−/− mice are resistant to LPS-induced lung inflammation. Wild-type and LPA₁^−/− mice were intratracheally challenged with LPS (5 mg/kg body wt) for 24 h. A: IL-6 levels in bronchoalveolar lavage fluids (BALF) were measured by ELISA. WT, wild type. B: lung tissues were stained with hematoxylin and eosin (H&E). Representative images are shown. C: cell counts in bronchoalveolar lavage (BAL) fluids were examined. D: the range of protein concentrations in BAL fluids and the means are shown. NS, not significant.

Fig. 7.

Inhibition of LPA receptors reduces LPS-induced lung inflammation in vivo. C57/BL6 wild-type mice were intratracheally challenged with DMSO (0.1% in H₂O) or Ki16425 (Ki, 5 μM in 25 μl H₂O) before LPS challenge (5 mg/kg body wt it, 24 h). A: IL-6 levels in BAL fluids were measured by ELISA. B: lung tissues were stained with H&E. Representative images are shown. C: cell counts in BAL fluids were examined. D: the range of protein concentrations in BAL fluids are shown along with the means.