Pulmonary epithelial CCR3 promotes LPS-induced lung inflammation by mediating release of IL-8

Pulmonary Epithelial CCR3Promotes LPS-Induced LungInflammation by MediatingRelease of IL-8BO LI,1,2,3 CHUNLING DONG,1,4 GUIFANGWANG,1 HUIRU ZHENG,1 XIANGDONGWANG,1*

AND CHUNXUE BAI1*1Department of Pulmonary Medicine, Institute of Respiratory Disease, Zhongshan Hospital, Fudan University, Shanghai, China2Department of Human Anatomy, Norman Bethune College of Medicine, Jilin University, Changchun, China3Laboratory of Physical Biology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai, China4Department of Respiratory Medicine, Second Hospital, Jilin University, Changchun, China

Interleukin (IL)-8 from pulmonary epithelial cells has been suggested to play an important role in the airway inflammation, although themechanism remains unclear. We envisioned a possibility that pulmonary epithelial CCR3 could be involved in secretion and regulation ofIL-8 and promote lipopolysaccharide (LPS)-induced lung inflammation. Human bronchial epithelial cell line NCI-H292 and alveolar type IIepithelial cell line A549 were used to test role of CCR3 in production of IL-8 at cellular level. In vivo studies were performed on C57/BL6mice instilled intratracheally with LPS in a model of acute lung injury (ALI). The activity of a CCR3-specific inhibitor (SB-328437) wasmeasured in both in vitro and in vivo systems.We found that expression of CCR3 inNCI-H292 and A549 cells were increased by 23% and16%, respectively, 24 h after the challenge with LPS. LPS increased the expression of CCR3 in NCI-H292 and A549 cells in a time-dependent manner, which was inhibited significantly by SB-328437. SB-328437 also diminished neutrophil recruitment in alveolarairspaces and improved LPS-induced ALI and production of IL-8 in bronchoalveolar lavage fluid. These results suggest that pulmonaryepithelial CCR3 be involved in progression of LPS-induced lung inflammation by mediating release of IL-8. CCR3 in pulmonary epitheliamay be an attractive target for development of therapies for ALI. J. Cell. Physiol.J. Cell. Physiol. 226: 2398–2405, 2011. � 2010 Wiley-Liss, Inc.

Acute lung injury (ALI) and acute respiratory distress syndrome(ARDS) are clinical syndromes characterized by an excessiveinflammatory response to both pulmonary and extrapulmonarystimuli, leading to a disruption of alveolar-capillary integritywithsevere consequences for pulmonary gas exchange (Ware andMatthay, 2000). Themortality rate of patients with ALI/ARDS isapproximately between 40% and 60% due to a poorunderstanding of potential mechanisms implicated in thepathogenesis of ALI/ARDS (Rubenfeld et al., 2005).Lipopolysaccharide (LPS) could induce ALI/ARDS in animalscharacterized by increased infiltration of inflammatory cells,production of inflammatory mediators, and tissue edema(Munoz et al., 2009).

Recruitment of neutrophil (polymorphonuclear leukocyte;PMN) into the lung is a key event in the early development ofALI/ARDS in animals and humans (Abraham et al., 2000;Azoulay et al., 2002) and occurs in a cascade-like sequence ofactivation, sequestration in pulmonary vessels, andtransendothelial (from blood to interstitium) andtransepithelial (from interstitium to alveolar airspace)migration (Reutershan et al., 2005). Each migration step couldbe regulated by distinct molecules including the chemokines(Zlotnik and Yoshie, 2000; Thelen, 2001; Razavi et al., 2004).Of them, the CC chemokine receptor CCR3 belongs to family1 of the G protein-coupled receptors binding and respondingto a variety of chemokines and highly expresses on multiplecells (Oliveira and Lukacs, 2003). Constitutive expressionof the CCR3 receptor was detected on normal humanbronchial epithelial cells, bronchial epithelial cell lines, andA549human alveolar type II epithelial cells (Stellato et al., 2001;Adachi et al., 2004; Heiman et al., 2005). CCR3 is involved in theaccumulation and activation of inflammatory cells (Oliveiraand Lukacs, 2003) and possess of wound repair, epithelialcell proliferation and chemotaxis, and the expression

of profibrogenic and chemokine transcripts (Beck et al.,2006).

Several studies have demonstrated the importance of thealveolar type II epithelial cells in the pathogenesis of andrecovery from severe ALI and ARDS (Geiser, 2003).Considering that each region of pulmonary epithelia mayrespond differently when exposed to inflammation-inducingagents, it is necessary to study the bronchial and alveolar type II

Bo Li and Chunling Dong contributed equally to this work.

Contract grant sponsor: Shanghai Leading Academic DisciplineProject;Contract grant number: B115.Contract grant sponsor: Fudan University.Contract grant sponsor: Shanghai Science and TechnologyCommittee;Contract grant number: 08PJ1402900, 08DZ2293104,9540702600.Contract grant sponsor: National Natural Science Foundation ofChina;Contract grant number: 30370611.Contract grant sponsor: Program for Outstanding MedicalAcademic Leader of Shanghai;Contract grant number: LJ06022.

*Correspondence to: Xiangdong Wang and Chunxue Bai,Department of Pulmonary Medicine, Zhongshan Hospital, FudanUniversity, 180 Fenglin Road, Shanghai 200032, China.E-mail: [email protected], [email protected]

Received 21 April 2010; Accepted 24 November 2010

Published online in Wiley Online Library(wileyonlinelibrary.com), 9 December 2010.DOI: 10.1002/jcp.22577

ORIGINAL RESEARCH ARTICLE 2398J o u r n a l o fJ o u r n a l o f

CellularPhysiologyCellularPhysiology

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epithelia together. We hypothesized that pulmonary epithelialCCR3 might be involved in production of interleukin-8 (IL-8),promoting LPS-induced lung inflammation. We found thatLPS-induced over-expression of CCR3 protein and mRNAin pulmonary epithelial cells and the CCR3-specific inhibitor(SB-328437) significantly decreased LPS-induced IL-8 secretionin NCL-H292 and A549 cells and the level of IL-8 in thebronchoalveolar lavage (BAL) fluid. SB-328437 effectivelydiminished neutrophils recruitment in alveolar airspaces andimproved LPS-induced ALI, indicating that pulmonary epithelialCCR3 were involved in LPS-induced lung inflammation bymediating release of IL-8.

Materials and MethodsCulture and stimulation of pulmonary epithelial cells

Human NCI-H292 bronchial epithelium-like cells and A549alveolar type II epithelium-like cells (ATCC CCL-185) werepurchased from American Type Culture Collection (Rockville,MD) and grown in RPMI-1640 medium supplemented with 10%fetal calf serum, penicillin (100U/ml), and streptomycin (100mg/ml)in a humidified atmosphere of 5% carbon dioxide at 378C. PBS/EDTA-dispersed cells were suspended in fresh medium in flasks orwells at 0.2� 106 or 1� 106 cells/ml, respectively. Experimentswere performed after subcultured cells had reached about 80%confluence. Viability of cells used for experiments was assessed bytrypan blue exclusion and the LIVE/DEAD viability-cytotoxicitycalcein AM/ethidium homodimer fluorescence assay (MolecularProbes, Eugene, OR). Only populations of cells with viability>93%were used for experiments. Prior to stimulation, cells wereincubated in serum-free medium for 24 h and then stimulated infresh serum-free medium with indicated concentrations of LPS(Sigma, St. Louis, MO).

Detection of pulmonary epithelial CCR3 by flow cytometry

NCI-H292 and A549 cells were treated with vehicle (medium) orLPS (10mg/ml) for 4, 12, and 24 h. Cells were detachedwith 0.5mMEDTA in PBS, centrifuged at 100g, 48C for 5min, washed twice incold PBS containing 0.5% BSA, and resuspended in PBS to a finalconcentration of 5� 106 cells/ml. Cells were then Fc-blocked bytreatment with 1mg human IgG/105 cells for 15min. Aliquots(25ml) of cells were stained with 10ml rabbit anti-human CCR3Polyclonal Ab (Lifespan biosciences, Seattle, WA) for 30min at48C. A FITC-conjugated goat anti-rabbit IgG was used as thesecondary Ab. After two washes in cold PBS, stained cells weremaintained at 48C then subjected to flow cytometry on aCytomicsTM FC 500 (Beckman Coulter, Fullerton, CA). Data wereanalyzed using CXP software.

Quantitative real-time PCR of CCR3 and IL-8

NCI-H292 and A549 cells were treated with the indicatedconcentrations of LPS for the indicated times. Cells were washedthree times with cold RPMI-1640 containing 10% fetal calf serum,lysed with Trizol (Life Technologies, Rockville, MD), and RNAextracted according to themanufacturer’s protocol. Before cDNAsynthesis, eachRNAsamplewas treatedwithDNase (Invitrogen byLife Technologies, Madison, WI) to eliminate any potentialcontamination with genomic DNA. Reverse transcription wasperformed on 2mg of total RNA/sample using Superscript II(RNase H-; Invitrogen Life Technologies) and oligo (dT) primerfrom SuperScript Choice system (Invitrogen Life Technologies) asthe first-strand cDNA primer. Real-time PCR analysis wasconducted in duplicate in the ABI PRISM 7900 SequencesDetection System. Negative controls were included in each assayto ensure that PCR product was not the result of reactioncontamination. The degree of change in transcript levels wascalculated relative to control group using the delta delta cyclethreshold (Ct) method (Kikuchi et al., 2004; Inoue et al., 2006). The

expression of housekeeping gene b-actin was used as thereference standard. Real-time PCR primers targeting CCR3, IL-8,and b-actin were designed using Primer Express software (AppliedBiosystems, Foster City, CA) with similar melting pointtemperatures, primer lengths and amplicon lengths to obtainsimilar PCRefficiency. The primerswere as follows:CCR3 forwardprimer: 50-AAAGCTGATACCAGAGCACTG-30, reverse primer:50-CCAAGAGGCCCACAGTGAAC-30. IL-8 forward primer:50-AGCTGGCCGTGGCTCTCT-30,reverse primer: 50-CCTTGGCAAAACTGCACCTT-30. b-Actinforward primer: 50-CTGGCACCCAGCACAATG-30, reverseprimer: 50-CCGATCCACACGGAGTACTTG-30.

Detection of IL-8 secretion in NCI-H292 and A549 cells byspecific ELISA

After stimulation of NCI-H292 and A549 cells for the indicatedtimes with the indicated concentrations of LPS, supernatants wereremoved and centrifuged at 100g for 5min at 48C. Resultingsupernatants ere immediately assessed for presence of IL-8 by acommercial, specific ELISA kit (R&D Systems, Minneapolis, MN) inaccordance with the manufacture’s instruction.

Animals

C57/BL6 male mice (n¼ 45) weighing 20� 2 g were purchasedfrom Fudan University Animal Center. The animal room wasmaintained at 228C with a daily light-dark cycle (06:00–18:00 hlight). Mice were fed standard mice chow and provided water adlibitum. All animal experiments were approved by the Animal CareCommittee of Fudan University.

Murine model of LPS-induced ALI

The general procedure was modified from previous experiments(Song et al., 2000). Briefly, mice were anesthetized with anintraperitoneal injection of 0.1ml pentobarbital sodium (50mg/kg;Sigma) and placed in a supine position head up on a board tilted at508. A midline incision was performed in the neck and the tracheawas exposed. LPS (Sigma) was dissolved in 0.9% saline atconcentrations of 2mg/ml and instilled to the trachea at a dosage of5mg/kg with a 29-gauge needle. After intratracheal instillation, themice were placed in a vertical position and rotated for 0.5–1min todistribute the instillation evenly within the lungs.

In vivo experimental protocol

Forty-five mice were randomly divided into three groups (n¼ 15,each group): Control group, LPS group and LPSþ SB group. Themice in LPSþ SB group were pretreated with an intratrachealinstillation of 5mg/kg SB328437 (2mg/ml). Then, the mice in bothLPS and LPSþ SB groups were instilled intratracheally with LPSaccording to the above protocol. Meanwhile, the mice in controlgroup received the same volume of 0.9% saline and manipulations.Mice of all three groups were terminated 24 h after intratrachealinstillation of LPS or saline.

Bronchoalveolar lavage

Mice (n¼ 5, each group) were sacrificed by receiving euthanasia.The mice were intubated with a 24-gauge cannula after theirtracheas were surgically isolated. The lungs were flushedwith 0.9%saline in 0.2-ml increments. The fluid recovery rate was 87� 2%.Fluid was centrifuged for 5m at 300g. The supernatant wasassessed for presence of IL-8 by a commercial, specific ELISA kit(R&D Systems) in accordance with the manufacture’s instruction.The pellet was resuspended in 1ml PBS with 1% BSA and 0.1%sodium azide, and a 10-ml aliquot was used to count cells. Cellviability was determined by Trypan Blue (Sigma, St. Louis, MO)exclusion. In addition, cytospun cells were prepared using acytocentrifuge (Academy ofMilitaryMedical Sciences) forWright’sstaining and differential cell counting.

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Lung morphology

Mice (n¼ 5, each group) were sacrificed by receiving euthanasia.The tracheas of mice were exposed and cannulated with PE-90tubing. After the chests were opened, the lungs were removedand filled with 10% buffered formalin at an airway pressure of20 cmH2O. Lung tissue was embedded in paraffin and 5mmsections were cut for hematoxylin and eosin staining. A lung injuryscoringmethodwas applied to quantify changes in lung architecturevisible by light microscopy. The degree of microscopic injury wasscored based on the following variables: Alveolar and interstitialedema, neutrophil infiltration, and hemorrhage. The severity ofinjury was graded for each variable: No injury¼ 0; injury to 25% ofthe field¼ 1; injury to 50% of the field¼ 2; injury to 75% of thefield¼ 3; and diffuse injury¼ 4 (Su et al., 2003). All samples wereanalyzed based on a scaled grading system by a pathologist whowasblinded to the experimental protocol and the region of sampling. Atotal of three slides from each lung sample were randomly

screened and the mean was taken as the representative values ofthe sample.

CCR3 immunohistochemistry

Paraffin-embedded sections (5mm) were stained for CCR3 usingthe avidin–biotin technique (Vector Laboratories, Burlingame, CA)as previously described (Reutershan et al., 2006). Briefly,deparaffinized and rehydrated sections were incubatedwith avidin,10% goat serum, and 0.5% fish skin gelatin oil for 1 h to blocknonspecific binding. After washing with PBS, rabbit anti-mouseCCR3was added (1mg/ml) and incubated overnight. Sectionswerethen washed and incubated with 5mg/ml biotinylated goat anti-rabbit IgG for 1 h, followed by avidin–biotin–peroxidase complexes(Vectastain Elite ABC kit; Vector Laboratories), washed with PBS,incubated with diaminobenzidine (DAB kit; Vector Laboratories)and counterstained with hematoxylin.

Fig. 1. LPSup-regulatedCCR3 receptors on the surfaceofNCI-H292 (A) andA549cells (B).NCI-H292andA549 cellswere treatedwith vehicle(medium) or LPS (10mg/ml) for 4, 12, and 24 h and analyzed by flow cytometry. a: Negative control. b: Unstimulated cells. c: LPS (10mg/ml, 4 h).d: LPS (10mg/ml, 12h). e: LPS (10mg/ml, 24 h). f: Unstimulated cells. Data presented are representative of three separate experiments eachconducted in duplicate.


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Fig. 2. LPS-induced mRNA expression of CCR3 and IL-8 in NCI-H292 and A549 cells. A,B: LPS-stimulated mRNA expression of CCR3in NCI-H292 and A549 cells was time dependent. Cells were treated only with LPS (10mg/ml) for 0, 4, 12, and 24 h. MValues that differedsignificantly from the 0h group at P<0.05. C,D: LPS up-regulated IL-8 mRNA expression in NCI-H292 and A549 cells in a concentration-dependent manner. Cells were treated with a range of LPS concentrations for 24 h. MValues that differed significantly from the unstimulatedcells at P<0.05. E,F: LPS up-regulated IL-8mRNA expression in NCI-H292 and A549 cells in a time-dependent manner. Cells were treated onlywithLPS (10mg/ml) for 0, 4, 12, and 24 h.All treated groupswere significantly different from their time-matched controls atP<0.05.The levels ofCCR3 and IL-8mRNA were detected by quantitative real-time PCR. The data presented are an average of three separate experiments eachconducted in triplicate.



Fig. 3. Kinetic analysis of LPS-induced IL-8 secretion from NCI-H292 and A549 cells and intervention by SB-328437. A,B: LPS-stimulatedsecretion of IL-8 was concentration dependent. Cells were treated with a range of LPS concentrations for 24 h. MValues that differedsignificantly from the unstimulated cells at P<0.05. C,D: LPS-stimulated secretion of IL-8 was time dependent. Cells were treated onlywithLPS (10mg/ml) for 4, 12, and24 h.All treatedgroupswere significantly different fromtheir time-matched controls atP<0.05. E,F: SB-328437down-regulatedLPS-induced IL-8 secretion inNCI-H292andA549cells.Cellswerepretreated for30minwith0–100nMSB-328437 in serum-freemedia followed by the stimulation of LPS (10mg/ml) for 24 h. The control group received no treatment. The SB group only received thepretreatment of 100nM SB-328437. MValues that differed significantly from the control group at P<0.05; #Values that differed significantlyfrom the LPS group at P<0.05. The secretion of IL-8 was evaluated by specific ELISA. The data presented are an average of three separateexperiments each conducted in duplicate.


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Lung wet/dry weight ratio

Mice (n¼ 5, each group) were sacrificed by receiving euthanasia.After that, the chests of mice were opened by a mediansternotomy, and the whole lungs were excised. The wet weight ofexsanguinated entire lungs was measured by an electronicalbalance, and then the lungs were dried in oven at 608C for 72 hbefore recording their dry weight. Finally, the wet-to-dry weightratio was calculated.

Data handling and analysis

Unless otherwise stated, experiments were conducted in triplicateand repeated on at least three separate occasions (flow cytometerexperiments were performed in duplicate on several differentoccasions). Unless otherwise stated, all data are expressed asmean� SEM with the mean of triplicates from one experimentserving as one observation. When indicated, one-way ANOVAfollowed by the Dunnett post-test, as appropriate, was applied toexperimental results to determine statistical significance (P< 0.05)between indicated groups.

ResultsLPS up-regulated protein and mRNA expression ofCCR3 in NCI-H292 and A549 cells

Figure 1a–f demonstrates constitutive expression of CCR3 andLPS-induced expression of CCR3 in NCI-H292 and A549 cells.The fluorescence intensity was found in 39.5% of unstimulatedNCI-H292 cells (Fig. 1Ab), indicative of constitutive CCR3expression in NCI-H292 cells, while 44.8%, 49.7%, and 62.8% 4,12, and 24 h after LPS challenge, respectively (Fig. 1Ac–e). 58%of unstimulated A549 cells exhibited CCR3-positive (Fig. 1Bb),while 64.8%, 68.4%, and 74.2% 4, 12, and 24 h after LPSchallenge, respectively (Fig. 1Bc–e). CCR3 mRNA expressionwas measured by quantitative real-time PCR on mRNAextracted from NCI-H292 and A549 cells treated with 10mg/ml LPS. LPS increased the CCR3 mRNA levels in a time-dependent manner from 0 to 24 h, as shown in Figure 2A,B. LPSup-regulated expression of CCR3 in NCI-H292 and A549 cellsat the protein and mRNA levels.

LPS stimulated a concentration- and time-dependentproduction of IL-8 in NCI-H292 and A549 cells

LPS induced an increased mRNA level IL-8 in NCI-H292 andA549 cells in a concentration-dependent manner (Fig. 2C,D)

and a closely related time-dependent manner (Fig. 2E,F). LPSincreased IL-8 production from NCI-H292 and A549 cells in aconcentration-dependent manner (Fig. 3A,B) and a closelyrelated time-dependent manner (Fig. 3C,D). In the culturedsupernatant of the LPS-treated cells, the concentration of IL-8significantly increased above the basal level 4 h after the onset oftreatment and continued to increase steadily, until the last timepoint at 24 h after the onset of treatment.

SB-328437 down-regulated LPS-induced IL-8 secretionin NCI-H292 and A549 cells

Although CCR3 expression changes following stimulation withLPS, it is till unknown if the LPS-induced change in CCR3expression affects IL-8 production. To explore this possibility,the NCI-H292 and A549 cells were pretreated with 0–100 nMCCR3 antagonist SB-328437 and then stimulated with 10mg/mlLPS. Pretreatment with SB-328437 inhibited LPS-induced IL-8production inNCI-H292 cells by 38% and 60% at 50 and 100 nMconcentrations, respectively (Fig. 3E). Pretreatment with SB-328437 could inhibit 17% and 46% LPS-induced IL-8 productionin A549 cells, respectively (Fig. 3F). It is therefore accurate tosuggest that pulmonary epithelial CCR3 receptors play a certainrole in LPS-induced IL-8 secretion. Collectively, these resultssuggest that blocking the CCR3 receptors with an antagonistprevents certain signal pathway related to the release of IL-8.

Effects of SB-328437 on LPS-induced ALI

The murine model of LPS-induced ALI was used and IL-8concentration in the BAL fluid was measured. The level of IL-8in the BAL fluid of the LPS group was significantly higher at 24 hthan that of the control group (P< 0.05), while treatment withSB-328437 significantly prevented LPS-induced increased levelof IL-8 at 24 h, but still higher than the control group (P< 0.05;Fig. 4A). The numbers of leukocytes and PMN in the BAL fluid ofthe LPS group were significantly increased at 24 h compared tothe control group (P< 0.05), while treatment with SB-328437 significantly prevented LPS-induced recruitment ofleukocytes and PMN in the BAL fluid, but still higher than thecontrol group (P< 0.05). PMNs were the predominant celltype in LPS-induced lung inflammation (Fig. 4B).

CCR3 immunohistochemistry was used to demonstrateCCR3 expression in pulmonary epithelial cells in vivo. As shownin Figure 5A, the immunohistochemical reactivity of CCR3 waslocalized to not only the bronchial but also alveolar epithelialcells. The alveoli had more protein-rich fluid and more PMN

Fig. 4. SB-328437 reduced LPS-induced IL-8 release (A) and neutrophil (PMN) recruitment (B) in BAL fluid. MValues that differed significantlyfrom the control group at P<0.05; #Values that differed significantly from the LPS group at P<0.05. NU 5 in each group. Values are presented asmeanWSEM.



infiltration 24 h after LPS challenge (Fig. 5C) than the controlgroup (Fig. 5B). Severe congestion and hemorrhage wasalso found in the LPS group. Those changes were inhibited bySB-328437 (Fig. 5D). The edema score, neutrophil infiltrationscore, and the hemorrhage score in the LPSþ SB-328437 groupwere significantly lower than the LPS group (P< 0.05; Fig. 5E).The lung wet/dry weight ratio in the LPS group significantlyhigher than controls (P< 0.05), while the intratrachealinstillation of SB-328437 significantly attenuated LPS-inducedlung edema at 24 h (P< 0.05; Fig. 5F). Blocking the pulmonaryepithelial CCR3 receptors with SB-328437 effectively

diminished neutrophil recruitment in alveolar airspaces andimproved LPS-induced lung inflammatory response.

Discussion

The pulmonary epithelia involve in inflammation-associatedconditions such as ALI, ARDS, chronic obstructive pulmonarydisease, and asthma (Borchers et al., 2009; Gabbert andNelson,2009; Kropski et al., 2009). The chemokines released by thepulmonary epithelia may induce the selective recruitment ofleukocytes and lead to the respective lung inflammation, as

Fig. 5. SB-328437 improved LPS-induced ALI. A: CCR3 immunohistochemistry. B–D: Histological changes of LPS-induced ALI (magnification400T, scale bar 50mm).B:Control group.C: LPSgroup.D: SBgroup. E:Histology score 24 hafterALI. MValues that differed significantly fromtheLPS group at P<0.05. F: Lung wet/dry weight ratio. MValues that differed significantly from the control group at P<0.05; #Values that differedsignificantly from the LPS group at P<0.05. NU 5 in each group. Values are presented as meanWSEM.


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important events in the progress of inflammation response.Weevaluated that pulmonary epithelial CCR3 could be involved inthe production of IL-8 and promote LPS-induced pulmonaryinflammation. Our results demonstrated that the pulmonaryepithelial CCR3 might mediate the LPS-induced release of IL-8derived from pulmonary epithelial cells, evidenced by the factthat inhibition of CCR3 diminished neutrophil recruitmentin alveolar airspaces and improved the LPS-induced lunginflammation in the murine model of ALI.

As the specific ligands ofCCR3, the eotaxinsCCL11,CCL24,and CCL26 are constitutively expressed in A549 cells, but onlyCCL24 is released from the unstimulated cells (Abonyo et al.,2005). CCL11 up-regulated mRNA and protein expression ofCCR3 in the airway epithelial cell line BEAS-2B (Beck et al.,2006), while CCL24 may be involved in downmodulation ofCCR3 in the unstimulated A549 cells (Abonyo et al., 2005).CCL26 reduced expression of CCR3 receptors by 30–40% inIL-4-treated A549 cells. In the present study, we found that LPScould increase CCR3 protein and mRNA levels in NCI-H292and A549 cells, probably associated cytokine production, e.g.,tumor necrosis factor (TNF)-a and IL-1b, from pulmonaryepithelial cells (Thorley et al., 2007). TNF-a and IL-1b couldinduce the release of CCL11 from pulmonary epithelial cellsduring the first 24 h of stimulation of LPS (Heiman et al., 2005).CCL11-CCR3 signal transduction pathways may be activatedresulted in the up-regulation of CCR3 protein and mRNAexpression in pulmonary epithelial cells (Beck et al., 2006).

IL-8 (CXCL8) is a member of the CXC chemokine family,which is secreted by variety of cell types in inflammation,associated with the development and outcome of ALI/ARDS(Kurdowska et al., 2001, 2002). IL-8 activates and recruits theneutrophils which produce numerous mediators, includingelastase, metalloprotease, and oxygen radicals, and promotelung inflammation and damage in the early development of ALI/ARDS. In the present study, we found that LPS stimulated aconcentration- and time-dependent production of IL-8 in NCI-H292 and A549 cells. Although CCR3mRNA and proteinexpression in NCI-H292 and A549 cells appeared to be up-regulated by LPS, the definite function of CCR3 in LPS-inducedlung inflammation is unknown. To confirm the possibility thatpulmonary epithelial CCR3may be related to the release of IL-8induced by LPS, a CCR3-specific inhibitor (SB-328437) wasused in both in vitro and in vivo systems. We found that SB-328437 significantly prevented over-production of IL-8 inducedby LPS in both in vitro and in vivo. These results indicate thatpulmonary epithelial CCR3 is involved in the mechanisms of IL-8 production, by which pulmonary epithelial CCR3 modulatesthe release of IL8, probably through CCL11–CCR3 signaltransduction pathway. It was reported that the protein levels ofIL-8 derived from airway epithelial cells was increased byCCL11 (Beck et al., 2006). LPS may induce the secretion ofCCL11 derived from pulmonary epithelial cells and up-regulatethe expression of pulmonary epithelial CCR3 and in turninteracted with CCR3 for the stimulation of IL-8 release inpulmonary epithelia.

Preventing the overproduction of IL-8 may attenuate lunginflammation (Nakamura et al., 1991). The pulmonary epithelialCCR3 promotes LPS-induced lung inflammation by the over-production of IL-8, evidenced by the reduction of neutrophilsrecruitment in alveolar airspace and the improvement of LPS-induced ALI after the treatment of SB-328437.

We also found thatCCR3 in the bronchial and alveolar type IIepithelia could modulate the release of IL-8 and promote lunginflammation induced by LPS, contributing to the developmentof ALI/ARDS. There is a great need of ALI/ARDS-specifictherapies to control acute lung inflammation and improve thesurvival rate of patients with ALI/ARDS. Pulmonary epithelialCCR3 could be one of the potential therapeutic targets forALI/ARDS.

Acknowledgments

This project was supported by Shanghai Leading AcademicDiscipline Project (B115), Fudan University (DistinguishedProfessor Grant), Shanghai Science and TechnologyCommittee (08PJ1402900, 08DZ2293104, 9540702600),National Natural Science Foundation of China (30370611), andProgram forOutstandingMedical Academic Leader of Shanghai(LJ06022).

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Pulmonary epithelial CCR3 promotes LPS-induced lung inflammation by mediating release of IL-8

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Transcript of Pulmonary epithelial CCR3 promotes LPS-induced lung inflammation by mediating release of IL-8