Phospholipase Cε plays a crucial role in neutrophilic inflammation accompanying acute lung injury through augmentation of CXC chemokine production from alveolar epithelial cells
We have shown that phospholipase Cε (PLCε), an effector of Ras and Rap1 small GTPases, plays pivotal roles in inflammation and inflammation-associated carcinogenesis by augmenting proinflammatory cytokine production from epithelial cells of various organs. The purpose of this study is to analyze its role in neutrophilic alveolar inflammation accompanying acute lung injury (ALI), focusing on that in alveolar epithelial cells (AECs), which are known to make a major contribution to the pathogenesis of ALI.
We examine the effect of the PLCε genotypes on the development of ALI induced by intratracheal administration of lipopolysaccharide (LPS) to PLCε wild-type (PLCε+/+) and knockout (PLCεΔX/ΔX) mice. Pathogenesis of ALI is analyzed by histological examination of lung inflammation and measurements of the levels of various cytokines, in particular neutrophil-attracting chemokines such as Cxcl5, by quantitative reverse transcription-polymerase chain reaction and immunostaining. Primary cultures of AECs, established from PLCε+/+ and PLCεΔX/ΔX mice, are used to analyze the roles of PLCε, protein kinase D (PKD) and nuclear factor-κB (NF-κB) in augmentation of LPS-induced Cxcl5 expression.
Compared to PLCε+/+ mice, PLCεΔX/ΔX mice exhibit marked alleviation of lung inflammation as shown by great reduction in lung wet/dry weight ratios, accumulation of inflammatory cells in the alveolar space and thickening of alveolar walls as well as the number of neutrophils and the protein concentration in bronchoalveolar lavage fluid. Also, LPS-induced expression of the CXC family of chemokines, in particular Cxcl5, is substantially diminished in the total lung and AECs of PLCεΔX/ΔX mice. Moreover, LPS-induced Cxcl5 expression in primary cultured AECs is markedly suppressed on the PLCεΔX/ΔX background (p < 0.05 versus PLCε+/+ AECs), which is accompanied by the reduction in phosphorylation of inhibitor κB (IκB), PKD and nuclear translocation of NF-κB p65. Also, it is suppressed by the treatment with inhibitors of PKD and IκB kinase, suggesting the involvement of the PLCε-PKD-IκB-NF-κB pathway.
PLCε-mediated augmentation of the production of the CXC family of chemokines, in particular Cxcl5, in AECs plays a crucial role in neutrophilic alveolar inflammation accompanying ALI, suggesting that PLCε may be a potential molecular target for the treatment of acute respiratory distress syndrome.
KeywordsPhospholipase Cε Acute lung injury Acute respiratory distress syndrome Alveolar epithelial cells Inflammation Mouse model Neutrophil Nuclear factor-κB Cxcl5
Alveolar epithelial cell
Acute lung injury
Acute respiratory distress syndrome
Bronchoalveolar lavage fluid
Chemokine C-C motif ligand
Chemokine C-X-C motif ligand
G protein-coupled receptor
Hematoxylin and eosin
Inhibitor κB kinase
Myeloid differentiation primary response gene 88
Phosphate- buffered saline
Protein kinase D
Prosurfactant protein C
Quantitative reverse transcription-polymerase chain reaction
Acute respiratory distress syndrome (ARDS) is a life threatening respiratory condition characterized by tachypnea, refractory hypoxemia and diffuse opacities on chest radiographs following sepsis, pneumonia and trauma . The mortality of ARDS patients is around 35–40%, however, effective protocols for its treatment remains to be worked out despite exhaustive investigation . The pathological development of ARDS is traditionally classified into 3 successive phases: exudative, proliferative and fibrotic . The exudative phase is characterized by immune-cell-mediated damage to alveolar epithelial cells (AECs) and endothelial cells and by the dysfunction of the alveolar capillaries. In the proliferative phase, the numbers of abnormal type II alveolar cells and inflammatory cells are increased, and, in the fibrotic phase, fibroblasts infiltrate and replace cells in the alveoli and alveolar ducts as fibrosis develops. The current view of ARDS suggests that these 3 phases are overlapping with each other  . Classically, the most characteristic feature of ARDS is inflammation with heavy infiltration of neutrophils, which is mediated by elevation of proinflammatory cytokines such as interleukin (IL)-1β, IL-6, IL-8 and tumor necrosis factor-α (TNF-α). Additionally, leukocyte recruitment is orchestrated bychemokines, in particular the neutrophil-attracting chemokines, C-X-C motif ligands (CXCLs) and C-C motif ligands (CCLs) [1, 4]. The injured AECs act as the main source of these chemokines, thereby making a great contribution to the pathogenesis of ARDS . In this study, we focuse on the role of Cxcl5, which is produced from AECs, different from myeloid cells which produce most chemokines and reported to play a key role in neutrophil activation during the pathogenesis of ARDS .
Phospholipase C (PLC) hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate inositol 1,4,5-triphosphate and diacylglycerol (DAG), which function as vital intracellular second messengers through mobilization of Ca2+ from the intracellular stores and activation of DAG-target proteins such as protein kinases C and D (PKD), respectively . Thirteen PLC isoforms are present in mammals and grouped into 6 classes: β, γ, δ, ε, ζ, and η, based on the similarities in their structures and regulatory mechanisms. Among them, PLCε is the only isoform regulated by small GTPases Ras and Rap , and further studies showed that it is also regulated by Rho small GTPases and heterotrimeric G protein Gα12, Gα13 and β1γ2 subunits . Stimulation of G protein-coupled receptors (GPCRs) with their ligands such as lysophosphatidic acid (LPA), sphingosine-1-phosphate (S1P) and thrombin induces PLCε activation . PLCε is expressed in structural cells such as epithelial cells and endothelial cells but not in immune cells such as granulocytes, lymphocytes and macrophages [10, 11, 12]. By utilizing genetically modified mice for PLCε, we demonstrated that PLCε plays a crucial role in experimentally induced inflammation; mice homozygous for the inactivated PLCεalleles (PLCεΔX/ΔX mice) exhibited markedly attenuated inflammatory responses in various animal models including the phorbor ester-induced dermatitis, hapten-induced contact dermatitis and dextran sulfate-induced colitis models [10, 11, 13] and transgenic mice overexpressing PLCε specifically in the skin keratinocytes spontaneously developed chronic dermatitis resembling human psoriasis . Concurrently, PLCεΔX/ΔX mice showed marked resistance to tumor formation in the two-stage skin chemical carcinogenesis and the de novo intestinal carcinogenesis on the APCMin/+ background, which were associated with attenuation of cancer-associated inflammation [15, 16]. In the lung, we found that PLCε plays a crucial role in Th2-cell-mediated eosinophilic inflammation in the mouse model of ovalbumin-sensitized allergic bronchial asthma . In this model, primary culture of bronchial epithelial cells was used to demonstrate that PLCε is required for TNF-α-induced pro-inflammatory cytokine production. Recently, we showed that, in human colon epithelial Caco2 cells, PLCε enhances pro-inflammatory cytokine expression through nuclear factor-κB (NF-κB) activation through activation of PKD, thereby recruiting and activating immune cells to initiate and sustain inflammation . It was shown that the PLCε-PKD axis, activated by LPA receptor engagement, augments cytokine production via NF-κB activation through phosphorylation and degradation of inhibitor κB (IκB) by ribosomal S6 kinase (RSK), not by IκB kinase (IKK) in the canonical NF-κB pathway.
Thus, we examine the role of PLCε in AECs in the pathogenesis of experimentally induced ARDS by employing the mouse model of LPS-induced ALI.
LPS from Escherichia coli O111:B4 (L2630, Sigma-Aldrich, St. Louis, MO, USA), IKK inhibitor BMS-345541 (401,480, Calbiochem, Darmstadt, Germany), PKD inhibitor CID755676 (476,495, Calbiochem) and proteasome inhibitor MG-132(Calbiochem) were commercially obtained. Primary antibodies against the following proteins were purchased from Cell Signaling Technology (Denver, MA, USA): β-actin (#4967), phospho-IκBα (Ser32) (14D4) (#2859), IκBα (#9242), phospho-PKD (Ser916) (#2051) and PKD (#2052). Other antibodies used were anti-CD45 (30-F11, eBioscience, San Diego, CA, USA), anti-CD32 (93, eBioscience), anti-Cxcl5 (PAA860Mu01, Cloud-Clone Corp, Katy, TX), anti-pan-cytokeratin (C-11, ab7753, Abcam, Cambridge, UK), anti-prosurfactant protein C (pro-SPC) (ab90716, Abcam), anti-podoplanin (bs-1048R, Bioss, Boston, MA, USA) and anti-NF-κB p65 (sc-109, Santa Cruz, Dallas, TX, USA). In addition, CF dye-labeled secondary antibodies, CF488A (20015) and CF555 (20231), were purchased from Biotium (Hayward, CA, USA).
PLCεΔX/ΔX mice homozygous for the allele harboring an in-frame deletion of the catalytic X domain of PLCε and hence devoid of its lipase activity  were backcrossed to C57BL6JJcl mice (CLEA Japan, Tokyo, Japan) for at least 8 generations. All animals were maintained in the animal facility of Kobe University Graduate School of Medicine. The use and care of animals were reviewed and approved by the Institutional Animal Care and Use Committee of Kobe University (Permit Numbers: P130612, P130612-R1 and P130401).
Primary culture of type II AECs was prepared from adult naïve PLCε+/+ and PLCεΔX/ΔX mice according to the previously described protocol [17, 18, 19]. Culture dishes (Φ = 10 cm) coated with anti-mouse CD45 antibody (40 μg) and anti-mouse CD32 antibody (15 μg) were used to get rid of leukocytes from the culture. The epithelial nature of the cultured cells was confirmed by positive pan-cytokeratin staining in at least 97% of the cells. They were further checked by positive staining for pro-SPC and podoplanin, specific markers of AECs (Additional file 1: Figure S1). On day 3, the cells were stimulated with LPS (500 ng/mL) for various times [6, 20] and used for further analyses. When necessary, the cells were treated with the IKK inhibitor (20 μM) for 30 min or with the PKD inhibitor (10 μg/ml) for 60 min before LPS stimulation.
Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
Preparation of total cellular RNAs and qRT-PCR using them as a template were performed as described previously . The primers used for the qRT-PCR analyses of various chemokine mRNAs are summarized in Additional file 2: Table S1. The relative mRNA levels were calculated according to the ΔΔCt method using the β-actin mRNA as an internal control.
Western immunoblotting and immunofluorescence staining analyses for cytokines expressed in AECs
Cultured AECs were lysed in Cell Lysis Buffer (Cell Signaling Technology) and total cellular proteins (20 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by Western immunoblotting as described before . Immunofluorescence staining was performed as previously described with minor modifications .
Induction of ALI by LPS administration
PLCε+/+ and PLCεΔX/ΔX mice at 8–10 weeks of age were anesthetized by intraperitoneal injection of dexmedetomidine (40 μl; Maruishi Pharmaceutical, Osaka, Japan), midazolam (11 μl; Astellas Pharma, Tokyo, Japan) and butorphanol tartrate (13 μl; Meiji Seika Pharma, Tokyo, Japan). The mice were randomly divided into the ALI model group and the control group, and received intratracheal administration of 50 μl phosphate-buffered saline (PBS) with and without LPS (5 mg/kg), respectively . Twenty-four hours later, the mice were sacrificed for dissection of the lungs or subjected to the collection of bronchoalveolar lavage fluid (BALF).
Collection and analysis of BALF
BALF samples were collected by washing the lung three times with 0.8 ml PBS through a tracheal cannula placed into each mouse under anesthesia as described previously . The number of leukocytes in the BALF was counted using a hemocytometer. The BALF was then centrifuged at 1500 × rpm for 10 min at 4 °C, and the cell pellet was suspended in 50 μl PBS, subjected to cytospin preparation and stained by Romanowski protocol using Diff-Quik (Sysmex, Kobe, Japan). At least 200 leukocytes on each slide were subjected to differential counting of macrophages, neutrophils, lymphocytes and eosinophils according to the standard morphological criteria.
Protein and cytokine measurements in BALF
The concentration of total protein in BALF was measured by using the Bradford protein assay (Bio-Rad, Hercules, CA, USA). The concentration of Cxcl5 in BALF was determined using the ELISA kit (R&D systems, Minneapolis, MN, USA).
Histopathologic and imunohistochemical analyses
The dissected lungs were filled with phosphate-buffered paraformaldehyde via intratracheal instillation and further fixed in phosphate-buffered paraformaldehyde. The lung tissues were embedded in paraffin, sectioned and subjected to staining with hematoxylin and eosin (H&E). The paraffin-embedded sections were also subjected to immunohistochemical staining with the anti-Cxcl5 antibody (15 μg/ml) and the anti-pro-SPC antibody (1 μg/ml) along with the peroxidase polymer anti-rabbit IgG reagents by using the Polymer method with ImmPRESS reagent (Vector Laboratories, Burlingame, CA, USA).
Measurements of the lung wet/dry weight ratio
The dissected lung was immediately weighed to measure the wet weight. Subsequently, it was dried in an incubator at 60 °C for 24 h until its weight was stabilized and was subjected to the measurement of the dry weight. The wet/dry weight ratio was calculated by dividing the wet weight by the dry weight.
Data are presented as the mean ± standard deviation (SD). Statistical analyses were performed using the Student’s unpaired t-test to compare the differences between two groups. If a p-value was smaller than 0.05, the difference was considered statistically significant.
Alleviation of ALI in PLCε ∆X/∆X mice
Crucial role of PLCε in chemokine production in AECs accompanying the LPS-induced ALI
Crucial role of PLCε in chemokine production in cultured AECs
Crucial role of the PLCε-PKD-NF-κB signaling in LPS-induced Cxcl5 expression
We demonstrated that PLCε plays a crucial role in the development of LPS-induced experimental ALI. By employing a similar experimental ALI model, Bijli et al. had reported a crucial role of PLCε in mediating endothelial cell inflammation and barrier disruption . In the present study, we focused on the role of PLCε in AECs, which had been shown to act as the main source of pro-inflammatory cytokines and chemokines and thereby play a key role in the pathogenesis and resolution of ALI through regulation of neutrophil influx . Also, AECs are known to play a pivotal role in innate immunity associated with the expression of TLR2 and TLR4 [28, 29]. TLR4 is a major receptor of LPS, and the LPS/TLR4 signaling is relayed by activation of the MyD88-dependent and MyD88-independent pathways, inducing the mRNA expression of pro-inflammatory cytokines and type I interferon [30, 31].
Our results using the lung tissues and primary AEC cultures from PLCε+/+ and PLCεΔX/ΔX mice suggested that PLCε might facilitate LPS-induced neutrophilic inflammation through augmentation of the production of the CXC family of chemokines, in particular Cxcl5, from AECs. Among the LPS-induced proinflammatory cytokines, we focused our study on chemokines that attract and activate neutrophils, in particular the CXC chemokines possessing the CXC cysteine motif . The CXC chemokines are divided into two subgroups depending on the presence or absence of the Glu-Leu-Arg (ELR) motif. The CXC chemokines with the ELR motif, exemplified by Cxcl1, Cxcl2, Cxcl5 and Cxcl15, induce neutrophil chemotaxis, and Cxcl1, Cxcl2, Cxcl5 and Cxcl15 had been reported to function as important neutrophil chemo-attractants associated with lung inflammation in mice [32, 33]. Among them, we focused on Cxcl5 because it is produced mainly by AECs and because it is known to induce neutrophil trafficking in response to LPS exposure in the lung . A key role of Cxcl5 had been shown in lung inflammation such as that occurred in the mouse model of pneumococcal pneumonia [34, 35]. Moreover, it had been reported that neutralization of Cxcl5 in the lung suppressed neutrophilic inflammation in a mouse model of LPS-induced ALI . Furthermore, it had been observed that the levels of Cxcl5 in BALF of patients with ARDS were high [36, 37, 38]. These findings strongly supported our notion that the PLCε-mediated production of Cxcl5 from AECs might be causal to the pathogenesis of the LPS-induced ALI.
It is known that the expression of CXC chemokines including Cxcl5 is regulated by NF-κB [39, 40]. The expression of Cxcl5 accompanying pneumococcal pneumonia had been shown to be induced by NF-κB activation . Here we showed that PLCε augments LPS-induced Cxcl5 expression through NF-κB activation via the PLCε-PKD-IκB pathway in AECs. This pathway had been demonstrated to be responsible for the late phase of NF-κB activation upon stimulation of cultured human colon epithelial cells by TNF-α, while the early phase was mediated by the canonical pathway using IKK for IκB phosphorylation and linked to the late phase by LPA . In AECs, it is likely that the link between the early and late phases is provided by S1P, which was known to induce PLCε activation  and cooperate with the TLR4 signaling to enhance cytokine production in human gingival epithelial cells  through engagement of its GPCR receptors. The importance of the PLCε-PKD-IκB pathway in mediating NF-κB activation downstream of TLRs had been suggested by the observations that PKD was essential for the MyD88-dependent TLR signaling  and that PKD was involved in chemokine release induced by TLR2, TLR4 and TLR5 . In our present study, the evidence for the critical role of the PLCε-PKD-IκB pathway came from the observation that the LPS-induced Cxcl5 expression was suppressed by the inhibitors of IKK and PKD and that the LPS-induced nuclear translocation of NF-κB and phosphorylation of PKD and IκB were suppressed in PLCε∆X/∆X AECs. The IKK inhibitor was expected to suppress both the early and late phases of NF-κB activation while the PKD inhibitor was expected to suppress only the late phase. Our present observation that the PKD inhibitors resulted in substantial inhibition of LPS-induced Cxcl5 expression suggested that the PLCε-PKD-IκB pathway played a major role in the late phase NF-κB activation.
The results, taken together, indicated that PLCε plays a crucial role in neutrophilic inflammation accompanying LPS-induced experimental ALI through augmentation of Cxcl5 production in AECs via activation of the PLCε-PKD-IκB-NF-κB pathway. This suggested that PLCε might become a promising candidate target for the treatment of ARDS, which was also supported by the fact that PLCε∆X/∆X mice were born and grew normally except for the development of mild abnormality in the heart valves  predicting the low toxicity of PLCε-specific inhibitors.
We thank Dr. Hironori Edamatsu of the Division of Molecular Biology and all the members of the Division of Respiratory Medicine, Kobe University Graduate School of Medicine, for their helpful discussions.
This work was supported by JSPS KAKENHI 26461188 to Kazuyuki Kobayashi.
Availability of data and materials
All data generated or analyzed during this study are included in this article.
KU and TN participated in study design. KU, TN, KK and TK wrote the manuscript. All authors analyzed the data and KK conceived the study. MY conducted the statistical analysis. TK and YN assisted with technical advice. All authors read and approved the final manuscript.
All the animal experiments were approved by the Institutional Animal Care and Use Committee (Permit Numbers: P130612, P130612-R1, and P130401) and carried out in accordance with the Kobe University Animal Experimentation Regulations.
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