&get_box_var;ORIGINAL ARTICLE

Interleukin-26 in Antibacterial Host Defense of Human LungsEffects on Neutrophil MobilizationKarlhans F. Che1, Sara Tengvall2*, Bettina Levanen1*, Elin Silverpil1, Margaretha E. Smith2, Muhammed Awad3,4,Max Vikstrom5, Lena Palmberg1, Ingemar Qvarfordt2, Magnus Skold3,4, and Anders Linden1,2,4

1Unit for Lung and Airway Research, Institute of Environmental Medicine, 3Unit of Respiratory Medicine, Department of MedicineSolna, 4Lung Allergy Clinic, and 5Unit for Cardiovascular Epidemiology, Institute of Environmental Medicine, Karolinska Institutet,Stockholm, Sweden; and 2Lung Immunology Group, Institute of Medicine, Sahlgrenska Academy at the University of Gothenburg,Gothenburg, Sweden

Abstract

Rationale: The role of the presumed Th17 cytokine IL-26 inantibacterial host defense of the lungs is not known.

Objectives: To characterize the role of IL-26 in antibacterial hostdefense of human lungs.

Methods: Intrabronchial exposure of healthy volunteers to endotoxinand vehicle was performed during bronchoscopy and bronchoalveolarlavage (BAL) samples were harvested. Intracellular IL-26 was detectedusing immunocytochemistry and immunocytofluorescence. This IL-26was also detected using flow cytometry, as was its receptor complex.Cytokines and phosphorylated signal transducer and activator oftranscription (STAT) 1 plus STAT3 were quantified using ELISA.Gene expression was analyzed by real-time polymerase chainreaction and neutrophil migration was assessed in vitro.

Measurements and Main Results: Extracellular IL-26 wasdetected in BAL samples without prior exposure in vivo and was

markedly increased after endotoxin exposure. Alveolarmacrophagesdisplayed gene expression for, contained, and released IL-26. Th andcytotoxic T cells also contained IL-26. In the BAL samples, IL-26concentrations and innate effector cells displayed a correlation.Recombinant IL-26 potentiated neutrophil chemotaxis inducedby IL-8 and fMLP but decreased chemokinesis for neutrophils.Myeloperoxidase in conditioned media from neutrophils wasdecreased. The IL-26 receptor complex was detected in neutrophilsand IL-26 decreased phosphorylated STAT3 in these cells. In BALand bronchial epithelial cells, IL-26 increased gene expression ofthe IL-26 receptor complex and STAT1 plus STAT3. Finally, IL-26increased the release of neutrophil-mobilizing cytokines in BALbut not in epithelial cells.

Conclusions:This study implies that alveolarmacrophages produceIL-26, which stimulates receptors on neutrophils and focuses theirmobilization toward bacteria and accumulated immune cells inhuman lungs.

Keywords: IL-10; infection; inflammation; macrophage; T cell

Bacterial infections in the lungs and airwaysaffect and kill millions of people worldwideeach year (1). Despite these facts andextensive research efforts, there is limitedunderstanding of the immunologicmechanisms underlying antibacterial host

defense of the lungs. This is unfortunategiven the increasing need for new therapythat can complement antibiotics or targetchronic inflammatory disorders.

There is evidence that Th17 cells arecritically involved in antibacterial host

defense of the lungs (2–10) and IL-26 isa relatively unknown cytokine that maybe produced by Th17 cells (11–14). Byreleasing their archetype cytokine IL-17A,these Th17 cells can contribute to therecruitment and accumulation of

(Received in original form April 16, 2014; accepted in final form October 2, 2014 )

*These authors contributed equally.

Supported by the Heart-Lung Fund (#20130294) and by King Gustav V’s and Queen Victoria’s Freemason Research Foundation. Federal funding wasobtained from Karolinska Institutet and, in accordance with the ALF/LUA agreement, from Stockholms Lans Landsting and the Vastra Gotaland Region.

Author Contributions: A.L. conceived the project, provided funding, and coordinated the research. K.F.C., S.T., B.L., E.S., M.E.S., L.P., I.Q., and A.L. outlined theresearch protocols. K.F.C., S.T., B.L., E.S., M.E.S., M.A., and M.S. performed the practical research. K.F.C., M.E.S., M.A., M.S., and A.L. provided humanspecimens. K.F.C., S.T., B.L., E.S., M.V., and A.L. reviewed the data. K.F.C. and A.L. wrote the manuscript. S.T., B.L., E.S., L.P., and I.Q. critically reviewed thecontents of the manuscript. All authors approved the final version of the manuscript.

Correspondence and requests for reprints should be addressed to Anders Linden, M.D., Ph.D., Unit for Lung and Airway Research, Physiology Division,Institute for Environmental Medicine, Karolinska Institutet, PO Box 210, SE 171 77 Stockholm, Sweden. E-mail: anders.linden@ki.se

This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org

Am J Respir Crit Care Med Vol 190, Iss 9, pp 1022–1031, Nov 1, 2014

Copyright © 2014 by the American Thoracic Society

Originally Published in Press as DOI: 10.1164/rccm.201404-0689OC on October 7, 2014

Internet address: www.atsjournals.org

1022 American Journal of Respiratory and Critical Care Medicine Volume 190 Number 9 | November 1 2014

neutrophils and macrophages duringinfections in the lungs (2, 7). However,given the variety of cytokines released byTh17 cells, remarkably little is known aboutthe corresponding role of Th17 cytokinesother than IL-17A. This is particularly true forIL-26, presumably because there is no knownhomolog to IL-26 in nonprimates (15).

In contrast to the lack of informationon antibacterial host defense in the lungs,evidence from human joint and gut tissuecells suggests that IL-26 is involved inchronic inflammatory disorders, such asrheumatoid arthritis (RA) (16) and Crohndisease (17). In a model of human liningepithelial cells from the colon and skin,recombinant human (rh) IL-26 stimulatedthe release of the neutrophil-recruitingC-X-C chemokine IL-8 (14) and was alsodemonstrated in gut tissue from patientswith Crohn’s disease (17). In support ofthese observations, the increased mRNA forIL-26 in Crohn’s disease correlated withmRNA for IL-8 (17) and the antibacterialTh17 cytokine IL-22 (17). Moreover, IL-26was markedly increased in synovial fluidfrom patients with RA and rhIL-26 inducedthe release of the neutrophil-mobilizingcytokines IL-1b, IL-6, tumor necrosis factor(TNF)-a, and IL-8 in human monocytes(16). Although this evidence is indirect, itis compatible with a proinflammatoryrole for IL-26 in antibacterial hostdefense (11, 15, 18).

Given that IL-26 and IL-17A can becoproduced by Th17 cells and that these

cells are present in human airways exposedto bacterial stimuli (2, 7, 10, 11), wehypothesized that IL-26, just like IL-17A,is involved and plays a critical role inantibacterial host defense of human lungs.We report here on evidence from healthyvolunteers and human primary cellssupporting this hypothesis. None of theresults in this study have previously beenreported in the form of an abstract.

Methods

We performed bronchoscopies andcollected bronchoalveolar lavage (BAL)samples in healthy volunteers, with orwithout intrabronchial exposure toendotoxin and vehicle (2, 19–21). With theaim to detect intracellular IL-26 in BALcells, we performed immunocytochemistry(ICC) and immunocytofluorescence (ICF)and fluorescence-activated cell sorter(FACS) (2, 19, 21). We also cultured andstimulated BAL cells, and isolated, cultured,and stimulated blood neutrophils andprimary bronchial epithelial cells (PBECs)(22). Moreover, we quantified cytokinesincluding IL-26 and phosphorylated signaltransducer and activator of transcription(STAT) 1 and STAT3 using ELISA,and gene expressions using real-timepolymerase chain reaction. Finally, we useda neutrophil migration assay (23). All themethods are described in the METHODS

section of the online supplement.

Statistical AnalysisParametric analysis was performed usingStudent paired t test (GraphPad Prismsoftware, GraphPad Software, Inc., SanDiego, CA) unless otherwise stated (see theMETHODS section in the online supplement).

Results

The Release of IL-26 in Response toEndotoxin and Its Association withNeutrophils and MacrophagesBecause local exposure to endotoxin in vivoincreases IL-17A and Th17-like cells inhealthy human airways (2), we determinedwhether IL-26 responds in a similarmanner. We used an established protocolfor unilateral, intrabronchial exposure inhealthy volunteers, allowing simultaneouscontrol sampling in a contralateralbronchial segment (2, 20). Bronchoscopieswere performed at the time of exposure and

were repeated 12, 24, or 48 hours later.We detected IL-26 in nonconcentrated,cell-free BAL fluid, even in BAL fluid fromthe vehicle-exposed bronchial segments,and these concentrations of IL-26 weremarkedly increased in the endotoxin-exposed segments of the same subjects.This pattern was observed in 30 out of the31 (97%) volunteers studied (Figure 1A).Moreover, endotoxin exposure in vitro ofBAL cells from healthy volunteers who hadnot been exposed to endotoxin or vehiclein vivo displayed a clear increase of IL-26concentrations in conditioned media(Figure 1B). In line with this, endotoxinexposure in vitro caused a 3.4-fold increasein IL-26 mRNA (Figure 1C). The BAL fluidfrom the same individuals (n = 18) alsocontained measurable IL-26 protein(135 [30–603] pg/ml), although theseconcentrations were somewhat lower thanthose in the BAL fluid from bronchialsegments exposed to vehicle. This wasprobably because of the absence ofa preceding bronchoscopy or spill-overof endotoxin from adjacently exposedbronchial segments (19, 24). Importantly,we found a strong correlation for IL-26 andneutrophils (Figure 1D) and macrophages(Figure 1E), but not for lymphocytes(Figure 1F) in these subjects. Notably, inBAL samples from volunteers undergoingunilateral, intrabronchial endotoxinexposure in vivo, increased IL-26 coincidedwith increased concentrations ofaccumulated innate immune cells, inparticular neutrophils, at 12, 24, or48 hours (cell counts published in Gladerand coworkers [2]). Using the Bernard test(see the METHODS section in the onlinesupplement), taking into account the useof two samples from each individual(i.e., vehicle- and endotoxin-exposedbronchial segments) as a single variable,we found direct associations of IL-26 andneutrophil (P = 0.002) and macrophage(P = 0.006) concentrations.

Intracellular IL-26 Protein in Small andLarge Mononuclear BAL CellsTh cells from human blood constitutesources of IL-26 that is coproduced with IL-17A and IL-22, designating Th17 cells asa source of IL-26 (11–14). However,Corvaisier and coworkers (16) recentlypublished evidence that IL-26 is secreted byCD681 synoviocytes in the joints ofpatients with RA. It is also known that thearchetype Th17 cytokine IL-17A can be

At a Glance Commentary

Scientific Knowledge on theSubject: The role of the presumedTh17 cytokine IL-26 in antibacterialhost defense of the lungs is not known.

What This Study Adds to theField: This study suggests that IL-26 isproduced by alveolar macrophagesmainly and exerts critical actions inantibacterial host defense of humanlungs. By stimulating receptors onneutrophils and by acting on othercells, IL-26 focuses neutrophilmobilization toward bacteria andaccumulated immune cells. Therefore,targeting IL-26 in severe infections andinflammatory disorders of the lungsmay have therapeutic potential.

ORIGINAL ARTICLE

Che, Tengvall, Levanen, et al.: IL-26 in Pulmonary Host Defense 1023

produced by CD681 alveolar macrophages(AM) and cytotoxic T (Tc) cells undercertain conditions, suggesting severalsources of IL-26 (25, 26). Consequently, ourinitial aim was to identify sources of IL-26among small and large mononuclear cellsfrom the airways. First, we analyzedunsorted BAL cells from healthy volunteersundergoing intrabronchial endotoxin-

exposure in vivo with respect to detectingIL-26 protein using specific monoclonalantibodies for ICC and ICF. The strongimmunoreactivity of the ICC indicated IL-26 protein in small and large mononuclearcells as opposed to the isotype control(Figures 2A and 2B). Our protocol clearlydiscriminated between positive andnegative cells (Figure 2B), arguing against

unspecific binding. To strengthen theevidence for IL-26 protein in the largemononuclear cells presumed to be AM,a specific monoclonal anti-CD68 antibodywas added. We then showed colocalizationof CD68 and IL-26 in large mononuclearBAL cells (Figure 2D), which was not thecase for the corresponding isotype controlantibody (Figure 2C). We confirmed thecolocalization of CD68 and IL-26 in thelarge mononuclear BAL cells using ICF(Figures 2E–2G).

Expression of IL-26 Protein inCytotoxic T Cells, T-Helper Cells, andMacrophagesUsing FACS, we next characterized the smallmononuclear sources of IL-26 in unsortedBAL cells from healthy volunteers that hadnot undergone prior in vivo exposure tovehicle and endotoxin. The BAL cells werestimulated ex vivo with 100 ng/ml ofendotoxin or vehicle during 24 hours, in theabsence of the protein transport inhibitormonensin. Here, we used specificmonoclonal antibodies against IL-26 (16)and against markers of T (CD3), Th (CD4),Tc (CD8) cells, and the Th17 transcriptionfactor (RORCvar2), plus the correspondingisotype control antibodies. We detectedcolocalization of IL-26 protein with all theseT-cell markers, without substantialdifferences between cells stimulated withvehicle or endotoxin (n = 6) (Figure 3A). Incontrast, we were unable to detect anycolocalization of IL-26 and IL-17A in thegated small mononuclear cells. This negativeoutcome is arguably conclusive because thesame FACS protocol clearly detected IL-17Ain Th17 cells differentiated from humanblood in vitro (data not shown). For thepurpose of optimizing the detection of IL-26–producing cells, we stimulated the BALcells during 4 hours with endotoxin orvehicle in the presence of monensin (n = 3).Again, we found colocalization of IL-26 withthe macrophage marker CD68 (41.3 [21–63]%) and with the T-cell markers CD3 (34.5[19.3–54.4] %), CD4 (27.7 [19.5–45.8] %),and CD8 (11.2 [7.58–14.6] %) (Figure 3B).However, there were no clear differencesbetween cells stimulated with vehicle orendotoxin.

Given the detected colocalization ofIL-26 and CD68 using ICC, ICF, and FACSon unsorted BAL cells, we conducted FACSon BAL cells enriched through adhesionand stimulated ex vivo with 1 mg/ml ofendotoxin or vehicle during 20 hours

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Figure 1. Effects of the toll-like receptor-4 receptor agonist endotoxin on the release of IL-26 in theairways. (A) Concentrations of IL-26 protein in cell-free bronchoalveolar (BAL) fluid from healthyvolunteers measured using ELISA. The BAL samples were harvested during bronchoscopy eitherat 12 hours (n = 6), 24 hours (n = 17), or 48 hours (n = 8) after intrabronchial exposure in vivo toendotoxin (squares, see METHODS) and vehicle (circles) in each subject. (B and C) Unsorted BAL cellsfrom healthy volunteers without prior exposure to endotoxin or vehicle in vivo were stimulated withendotoxin (squares, 1 mg/ml) or vehicle (circles) ex vivo (24 h) and IL-26 protein quantified by ELISA incell-free conditioned media (B) and mRNA of IL-26 measured using reverse-transcriptase polymerasechain reaction (C). Data sets on mRNA (C) are presented as fold differences of the mRNA fromthe vehicle (n = 8). A and B show matched data (connected with thin lines) for stimulation withendotoxin and vehicle in each donor. The bold lines indicate the mean values, and theP values according to Student paired t test are presented (A and B). Correlation of neutrophil(D), macrophage (E), and lymphocyte (F) concentrations (3106 cells/ml) and IL-26 in BAL samplesfrom subject without prior exposure to endotoxin or vehicle (n = 18). (D–F) P values according tothe Pearson correlation test are presented.

ORIGINAL ARTICLE

1024 American Journal of Respiratory and Critical Care Medicine Volume 190 Number 9 | November 1 2014

without monensin. Almost all the adherentBAL cells expressed CD68, in the presenceof endotoxin (99.5 [99.3–99.7] %) or itsvehicle (99.3 [99.2–99.7] %) (n = 4),verifying an enriched AM population. Inthese CD681 cells, we found a highpercentage of inherent expression of IL-26in the vehicle-exposed cells, one that wasfurther enhanced after stimulation withendotoxin (Figure 3C). To ascertain thatAM also release IL-26, we quantified IL-26protein in the conditioned media. We thendetected IL-26 after vehicle exposure andthis IL-26 was increased after stimulationwith endotoxin (Figure 3D). Finally, theadherent BAL cells were analyzed for

mRNA expression of IL-26 (n = 4) and wefound that the detected mRNA tended beincreased after stimulation with endotoxin(see Figure E1 in the online supplement).

IL-26 Potentiates Chemotaxis butInhibits Chemokinesis of NeutrophilsThe statistically proved correlations betweenIL-26 and neutrophils in BAL samples(Figure 1D) indicated that IL-26 exertseffects on neutrophil migration; even moreso, because ICC and ICF indicated thatBAL neutrophils do not express IL-26(Figure 2). To evaluate whether IL-26 mayexert a direct or indirect effect onneutrophil migration, we examined the

impact of rhIL-26 on chemotaxis andchemokinesis of blood neutrophils in vitro.We found that rhIL-26 potentiatedchemotaxis induced by IL-8 and fMLP(Figures 4A and 4B). By assessing themaximum effect caused by rhIL-26, wefound that rhIL-26 increased IL-8–inducedmigration by 4.2-fold (1.3–17.1) (n = 8) andfMLP-induced migration by 11.6-fold(1.1–41.9) (n = 5), suggesting strongpotentiation of chemotaxis. In contrast,stimulation with rhIL-26 alone in untreatedneutrophils inhibited spontaneousmigration (i.e., chemokinesis) (Figure 4C).

Effect of IL-26 on PhosphorylatedSTAT1 and STAT3 in NeutrophilsPrevious studies have linked receptorstimulation with IL-26 to alterations inSTAT1 and STAT3 (11, 12, 14, 15). Whenwe tried to verify functional IL-26 receptorsignaling in isolated neutrophils, wefound that rhIL-26 exerted a reproducibledecrease in phosphorylated STAT3 (Figure4D). Stimulation with rhIL-26 had acorresponding effect after costimulationwith granulocyte colony–stimulating factor(G-CSF; a potent STAT3-activator [27])(Figure 4D). In contrast, duringcostimulation with IL-8 or fMLP, rhIL-26did not alter phosphorylated STAT3 ina reproducible manner (see Figure E2A).Moreover, we found no effect of rhIL-26alone on STAT1, nor after costimulationwith G-CSF, IL-8, or fMLP (see Figures E2Band E2C).

Expression of the IL-26 ReceptorProtein Complex on NeutrophilsWe also investigated the expression of IL-26receptor complex (IL-10R2 and IL-20R1) onisolated blood neutrophils in vitro. Wethen found inherent expression of bothreceptor subunits (Figures 4E–4H) butprestimulation with rhIL-26, LPS, IL-8, orfMLP did not alter this receptor expression(see Figures E2D and E2E)

IL-26 Stimulates the Release ofNeutrophil-Mobilizing Cytokines andUp-regulates Gene Expression of ItsReceptor Complex and SignalingMolecules in BAL CellsIts proved link to neutrophil mobilization inRA and Crohn disease and our novel findingthat IL-26 was increased in BAL fluidfrom healthy volunteers after endotoxinexposure in vivo motivated us tocharacterize the effects of IL-26 on the

Figure 2. Detection of IL-26 ex vivo in airway immune cells after intrabronchial endotoxin exposurein vivo. Cytospin preparations of unsorted bronchoalveolar lavage cells were prepared forimmunocytochemistry (A–D) or immunocytofluorescence (E–G). Specifically, immunocytochemistrypanels show immunoreactivity for (A) monoclonal IgG2b isotype control antibody, (B) monoclonal-specific IL-26 antibody (red-brown; 1, positively stained; arrow, negatively stained cells), (C)a monoclonal IgG2b isotype control antibody plus a monoclonal antibody against the macrophagemarker CD68 (brown), and (D) monoclonal antibody against the macrophage marker CD68 (brown)plus a monoclonal IL-26 antibody (purple). A and B, magnification 340. C and D, magnification 325.The immunocytofluorescence panels show immunoreactivity for (E) the monoclonal IgG2b isotypecontrol antibody plus a monoclonal antibody against the macrophage marker CD68 (red), (F and G)a monoclonal antibody against the macrophage marker CD68 (red) plus a monoclonal IL-26 antibody(green). E and F, magnification 325; G, magnification 363. Each panel represents a typical resultfrom one out of four subjects analyzed with each respective method.

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Che, Tengvall, Levanen, et al.: IL-26 in Pulmonary Host Defense 1025

release of four neutrophil-mobilizingcytokines in unsorted BAL cells ex vivo. Wethen found that stimulation with rhIL-26increased IL-8, IL-1b, TNF-a, andgranulocyte-macrophage colony–stimulatingfactor (GM-CSF) in conditioned media(Figures 5A–5D). We also characterized theeffects of IL-26 on gene expression of the IL-26 receptor complex and the downstreamsignaling molecules in unsorted BAL cells.We detected the targeted mRNA (P valuesfor targeted mRNA relative to the house-keeping gene b-actin) and a clear increase inIL-10R2 (P = 0.03), IL-20R1 (P = 0.0057),and STAT1 (P = 0.01) after stimulation withrhIL-26 (Figure 5E). For STAT3 (P = 0.1),there was a trend toward an IL-26–inducedincrease only (Figure 5E).

IL-26 Inhibits the Release ofMyeloperoxidase Protein inNeutrophilsNeutrophils are considered themain sourcesof myeloperoxidase (MPO) protein in theairways (28) but these cells are scarce inBAL samples from healthy nonsmokingvolunteers (29) (see Figure E3). Given thelatter, we collected unsorted BAL sampleswith a neutrophil fraction of at least 2% ofthe total cell number and stimulated thesecells with rhIL-26 or vehicle during 24hours in vitro. This stimulation decreasedMPO in conditioned media (Figure 5F).The same type of inhibitory effect wasobserved in isolated blood neutrophilsin vitro (Figure 5G).

IL-26 Inhibits the Release ofNeutrophil-Mobilizing Cytokines andUp-regulates Gene Expression of ItsReceptor Complex and SignalingMolecules in Bronchial Epithelial CellsBronchial epithelial cells play a critical rolein initiating inflammatory responses to localbacteria (30). We therefore assessed theeffects of IL-26 on the release of neutrophil-mobilizing cytokines using PBECs,submerged in culture in vitro. Here,stimulation with rhIL-26 decreased IL-8,IL-1b, TNF-a, and GM-CSF in conditionedmedia (Figures 6A–6D). We alsocharacterized the effects of IL-26 on geneexpression of the IL-26 receptor complexand the downstream signaling molecules.We detected the targeted mRNA (P valuesfor targeted mRNA relative to the house-keeping gene b-actin) and a clear increasein IL-10R2 (P = 0.03), IL-20R1 (P = 0.02),STAT1 (P = 0.03), and STAT3 (P = 0.02)

% IL

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Figure 3. Detection of IL-26 in airway immune cells stimulated with endotoxin or vehicle ex vivo. (A)Individual percentage immunoreactivity for the colocalization of IL-26 with cell markers CD8, RORCvar2,CD4, and CD3 in unsorted bronchoalveolar lavage (BAL) cells gated from small mononuclear cellsduring fluorescence-activated cell sorter analysis. The unsorted BAL cells were stimulated (24 h) withendotoxin (100 ng/ml) or vehicle ex vivo, without monensin (n = 6). (B) Representative plots out of threeexperiments for the colocalization of intracellular IL-26 and the cell markers CD3, CD4, CD8, and CD68in unsorted BAL cells gated from mononuclear cells. The unsorted BAL cells were stimulated withendotoxin (1 mg/ml) or vehicle ex vivo (4 h), in the presence of monensin. (C) Individual percentageimmunoreactivity for the colocalization of IL-26 and CD68 in adherent BAL cells. The adherent BAL cellswere stimulated with endotoxin (squares, 1 mg/ml) or vehicle (circles) ex vivo (20 h), without monensinand analyzed using fluorescence-activated cell sorter (n = 4). (D) Concentrations of IL-26 protein in cell-free conditioned media from cultures of adherent BAL cells. The adherent BAL cells were stimulatedwith endotoxin (squares, 1 mg/ml) or vehicle (circles) ex vivo (20 h) and IL-26 protein measured usingELISA (n = 5). Thin lines for graphs indicate matched samples from each subject and bold lines indicatemean values. The P values indicated (A, C, and D) are according to Student paired t test.

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1026 American Journal of Respiratory and Critical Care Medicine Volume 190 Number 9 | November 1 2014

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p=0.03 p=0.08 p=0.015 p=0.002

Figure 4. Effects of IL-26 on neutrophil migration, phosphorylated signal transducer and activator of transcription (STAT) 3, and IL-26 surface receptor proteincomplex. Fluorescent-labeled neutrophils were added on the upper surface of a polycarbonate filter in a chemotaxis plate and the chemoattractants were addedin the bottom wells as specified in the figure panels. Plates were incubated (1 h) and the fluorescence of emigrated cells measured using a fluorescent platereader. (A) Effects of recombinant human (rh) IL-26 on neutrophil migration toward IL-8 (n = 8), (B) effects of rhIL-26 on neutrophil migration toward fMLP (n = 5),and (C) effects of IL-26 alone on spontaneous neutrophil migration (n = 8). (D) Dose-dependent effects of rhIL-26 on concentrations of phosphorylated STAT3(n = 11). Three hundred thousand blood neutrophils were stimulated (20 min) with different concentrations of IL-26. Cells were lysed and phosphorylated STAT3measured in the cell lysates using Phosphor Tracer ELISA. (E–H) The expression of IL-26 membrane receptor complex proteins on neutrophils after culture(2 h) ex vivo (n = 6). Data show mean with SEM. The P values indicated are according to Student paired t test (on logarithmically transformed data for A–C).

ORIGINAL ARTICLE

Che, Tengvall, Levanen, et al.: IL-26 in Pulmonary Host Defense 1027

after stimulation by rhIL-26 (Figure 6E). Inaddition, we examined the effects ofIL-26 on antimicrobial peptides (LL-37,calprotectin, and b defensin-2) plus the

secretory leukocyte peptidase inhibitor inthe conditioned media from PBEC usingELISA. Here, although concentrations weredetectable, stimulation with rhIL-26 did not

increase secretory leukocyte peptidaseinhibitor and calprotectin (see Figure E4).The concentrations for LL-37 and bdefensin-2 were not detectable.

Discussion

We investigated BAL samples from healthyvolunteers after intrabronchial exposure toendotoxin and to its vehicle and without anyprior exposure in vivo. We found detectableconcentrations of IL-26 even in bronchialsegments without prior exposure or withexposure to vehicle only. These IL-26concentrations were markedly increasedin bronchial segments with prior exposureto the toll-like receptor (TLR)-4 agonistendotoxin. Moreover, the concentrations ofinnate effector cells and IL-26 correlated inbronchial segments exposed to endotoxinor vehicle and unexposed healthyvolunteers. Given the massive dilution ofthe bronchoepithelial lining fluid duringBAL, these relatively high concentrationsindicate a substantial and inducible releaseof IL-26 protein in human lungs.

To identify the potential sources ofIL-26 among immune cells in thebronchoalveolar space, we used ICC, ICF,and FACS. Notably, these three approachesall provided evidence that AM constitutea prominent source of IL-26 in the lungs.Moreover, stimulation with endotoxinex vivo significantly increased IL-26 in theCD681 adherent BAL cells, indicatinginduced production of IL-26 in theabundant AM, a novel finding in line withpreviously published data on IL-26expression in CD681 synoviocytes inpatients with RA (16). Expanding theevaluation of IL-26 production in AM, wedemonstrated mRNA and an endotoxin-induced increase in intracellular IL-26 andextracellular IL-26 in a 99% positivefraction of CD681 cells. Collectively, thesefindings prove that AMs can produce andrelease IL-26. Given these novel findingsand the previously published negative data onhuman monocytes and dendritic cells (31), wespeculate that, after extravasation, monocytesneed to mature into tissue macrophages todevelop the capacity to produce IL-26. Theimmunologic importance of AM and theirrole as the most abundant “immune barriercells” in the lungs warrant further study inthis respect.

In addition to previous studies of IL-26in T cells (11–14), we here forward novelevidence for its production in T cells from

IL-1

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BA

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Figure 5. Effects of rhIL-26 on cytokine release, IL-26–receptor gene expression in bronchoalveolarlavage (BAL) cells, and myeloperoxidase (MPO) release in BAL cells and blood neutrophils. UnsortedBAL cells from unexposed healthy volunteers were cultured and stimulated with rhIL-26 (100 ng/ml) orvehicle ex vivo (24 h). Protein concentrations of (A) IL-8, (B) IL-1b, (C) tumor necrosis factor (TNF)-a, and(D) granulocyte-macrophage colony–stimulating factor (GM-CSF) measured in cell-free conditionedmedium are shown (n = 9 for all). (E) Fold gene expression measured in BAL cells for IL-10R2, IL-20R1,STAT1, and STAT3 is presented (n = 8). (F) The protein concentration of MPO in BAL cell-freeconditioned medium (n = 6) is shown. (G) Protein concentrations of MPO in cell-free conditionedmedium of blood neutrophils stimulated with rhIL-26 (100 ng/ml) or vehicle (18 h) ex vivo (n = 9). Datashow mean with SEM. The P values for all panels indicated are according to Student paired t test. ForF alone, a one-tailed paired t test was performed given the one-way hypothesis. STAT = signaltransducer and activator of transcription.

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1028 American Journal of Respiratory and Critical Care Medicine Volume 190 Number 9 | November 1 2014

human lungs as well. We demonstrate thepresence of IL-26 in small mononuclearBAL cells in vivo and the specificcolocalization of IL-26 and the generic T-cellmarker CD3, the Th marker CD4, and the Tcmarker CD8. Interestingly, the detection ofcolocalization of IL-26 with the Tc markerCD8 is an entirely novel finding irrespective ofhuman organ. It is to be expected that wefound significant intracellular expression ofIL-26 protein in T cells without endotoxin-induced increase. This is because T cells havevery limited expression of TLR-4 comparedwith macrophages and do not normallyproduce cytokines or proliferate in responseto endotoxin (32, 33).

We did detect colocalization of IL-26with the archetype Th17 transcription factor

RORCvar2 but only in a modest 14% of theendotoxin-stimulated small mononuclearcells. It thus seems unlikely that this Th17population accounts for the larger part ofthe IL-26 protein detected in the BALsamples of the current study, given ourdemonstration of its transcription,production, and release by abundant AM.Thus, we suggest that IL-26 may beproduced by several types of immune cells inhealthy human lungs but AM emerge as themost prominent source during activationof antibacterial host defense, at least afterexposure to a TLR-4 agonist.

Although we were unable to blockendogenous IL-26 signaling in humansin vivo for ethical reasons, our current studybrings forward no less than five lines of

evidence that local IL-26 is functionallyimportant for the mobilization ofneutrophils to sites of bacteria andaccumulated immune cells in human lungs.First, the induced increase in IL-26 wasassociated with a pronounced accumulationof innate effector cells in vivo and theconcentrations of these entities evencorrelated with one another. Second, wefound that IL-26 potentiated neutrophilchemotaxis induced by IL-8 (z4-fold) orfMLP (z12-fold) in vitro. Third, we foundthat IL-26 alone inhibited chemokinesis ofneutrophils in vitro. Fourth, we establishedevidence for a functional IL-26 membraneprotein receptor complex in neutrophils byusing FACS and by showing an induceddecrease in phosphorylated STAT3 byrhIL-26. We found that IL-26 decreasedphosphorylated STAT3 even aftercostimulation with the potent STAT3-activator G-CSF but not in the presenceof IL-8 or fMLP, which is compatible withprevious studies (27). In this way, IL-26may serve an important immunologicpurpose, by inhibiting random migration ofneutrophils through the inhibition ofSTAT3. In contrast, IL-26 may promotemeaningful migration toward gradients ofchemokines (IL-8) and compounds frominvading bacteria (fMLP) via a mechanismseparate from STAT3, a potentiallyimportant area for future mechanisticresearch. Fifth, we identified stimulatingeffects of IL-26 on four archetypeneutrophil-mobilizing cytokines releasedfrom immune cells in the unsorted BALsamples cultured ex vivo, including IL-1b,TNF-a, GM-CSF, and IL-8.

It seems likely that AM, the mostabundant immune barrier cell in theairways, constitute the main source ofthese cytokines, given the publisheddemonstration that IL-26 increases therelease of proinflammatory cytokines inblood monocytes (16). Here, in terms ofneutrophil mobilization, it is alsointeresting to note that IL-8 may not onlyact as a chemokine, but also as an enhancerof neutrophil activity during infections ortissue injury (34, 35). We also find theinhibitory effect of IL-26 on the releaseof the very same neutrophil-mobilizingcytokines in PBEC cultured in vitrointriguing. Given the other four lines ofevidence, this particular finding points outthe possibility that IL-26 favors neutrophilmobilization where bacteria and immunecells are accumulated rather than in the

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E

Figure 6. Effects of rhIL-26 on cytokine release, and IL-26–receptor gene expression in structuralairway cells. Primary bronchial epithelial cells from unexposed human volunteers were stimulated withrhIL-26 (100 ng/ml) or vehicle in culture (24 h) ex vivo. Concentrations of (A) IL-8, (B) IL-1b, (C) tumornecrosis factor (TNF)-a, and (D) granulocyte-macrophage colony–stimulating factor (GM-CSF) incell-free conditioned medium (n = 9 for all groups). (E) Fold gene expression measured in primarybronchial epithelial cells for IL-10R2, IL-20R1, STAT1, and STAT3 (n = 6). Data show mean with SEM.The P values for all panels indicated are according to Student paired t test. STAT = signal transducerand activator of transcription.

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Che, Tengvall, Levanen, et al.: IL-26 in Pulmonary Host Defense 1029

airway mucosa per se. Future studies onorgans other than the lungs may show howgeneric are these proposed mechanisms

Another mechanistic link between IL-26 and neutrophil mobilization addressed inthis study is that IL-26 inhibits the inherentrelease of MPO, the neutrophil activitymarker (28). Hypothetically, this inhibitionby IL-26 may prevent MPO release untila bacterial stimulus is encountered. Clearly,this type of inhibitory effect by IL-26bears therapeutic potential for severecomplications to infections, including acutelung injury and other chronic inflammatorydisorders characterized by excessiveneutrophil activity (36).

We also examined the effect of rhIL-26on the gene expression of its own receptorsubunits IL-10R2 and IL-20R1 and of itsdownstream intracellular signalingmolecules STAT1 and STAT3 in BAL cellsand PBEC. We find it exciting that ourresults indicate a positive feedbackmechanism by which IL-26 can up-regulatethe gene expression of its receptor complex,and its downstream signal molecules, inimmune cells and structural cells of thelungs. Hypothetically, it is possible thatafter bacterial exposure in human lungs,IL-26 is increased and enhances the localresponsiveness to restimulation by itself.

It is of mechanistic interest that theopposing effects of IL-26 in immune and

structural cells are compatible with IL-26acting on more than one type of receptorcomplex, a possibility deserving furtherstudy. Moreover, IL-8, and to some extentGM-CSF, are of particular importance forthe mobilization of neutrophils duringbacterial infections but IL-1b and TNF-arepresent more “generic” proinflammatoryand pyrogenic cytokines (37–42). IL-1band TNF-a typically emanate frommacrophages and numerous immune andstructural cells during acute injury orchronic inflammatory disorders in thelungs, in addition to infections caused byextracellular bacteria (37–42). Even the IL-26–induced release of GM-CSF may havebearing for acute injury or chronicinflammatory disorders because this growthfactor is believed to be an importantsurvival and immune activation factor inresponse to tissue injuries in the lungs (43,44). Moreover, as indicated by publicationsemerging while the current manuscriptwas being written, IL-26 may be involved inadditional aspects of host defense, includinginfections by intracellular bacteria orviruses (45, 46).

In conclusion, this study forwardsoriginal evidence that IL-26 is involved andplays a critical role in antibacterial hostdefense of human lungs. The study suggeststhat IL-26 is abundantly produced andreleased by AM, possibly by local Th and Tc.

Our study shows that neutrophils havefunctional receptors for IL-26 and indicatesthat, as a net result of its immunologiceffects, IL-26 potentiates neutrophilmigration toward the bacterial compoundfMLP and the archetype chemokine IL-8.This implies that, in human lungs, IL-26focuses neutrophil mobilization toward sitesof bacteria and accumulated immune cellsreleasing chemokines. This intriguinginvolvement of IL-26 in antibacterial hostdefense deserves to be further studiedbecause it bears potential for therapy. n

Author disclosures are available with the textof this article at www.atsjournals.org.

Acknowledgment: The authors thank PernillaGlader, Ph.D., Marit Hansson, M.D., andLouise Bramer, B.Sci., for their methodologiccontribution during the preparation for this study.They also thank Professor Esbjorn Telemo for hisexpert advice during the preparation of thework with ICC and ICF. They thank KristinBlidberg, Ph.D., for assisting in setting up thechemotaxis assay. In addition, they thankProfessor Kjell Larsson for his critical commentson the manuscript. Moreover, they gratefullyacknowledge the research nurses HeleneBlomqvist, Gunnel de Forest, and Margitha Dahlat the Lung Allergy Clinic at Karolinska UniversityHospital Solna, and the research nurse BarbroBalder, at the Section for Respiratory Medicine,Sahlgrenska University Hospital, for theirimportant work in recruiting and examining thehealthy volunteers included in this study.

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