of April 11, 2012This information is current as

2008;181;4089-4097J Immunol KollsCharles G. Irvin, Jon D. Piganelli, Anuradha Ray and Jay K.DuPont, Shernaaz Kapadia, Alison Logar, Adam Henry, Laura McKinley, John F. Alcorn, Alanna Peterson, Rachel B. in MiceInflammation and Airway Hyperresponsiveness TH17 Cells Mediate Steroid-Resistant Airway

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TH17 Cells Mediate Steroid-Resistant Airway Inflammationand Airway Hyperresponsiveness in Mice1

Laura McKinley,* John F. Alcorn,* Alanna Peterson,* Rachel B. DuPont,* Shernaaz Kapadia,*Alison Logar,* Adam Henry,† Charles G. Irvin,‡ Jon D. Piganelli,* Anuradha Ray,†

and Jay K. Kolls2*

Steroid-resistant asthma comprises an important source of morbidity in patient populations. TH17 cells represent a distinctpopulation of CD4� Th cells that mediate neutrophilic inflammation and are characterized by the production of IL-17, IL-22, andIL-6. To investigate the function of TH17 cells in the context of Ag-induced airway inflammation, we polarized naive CD4� T cellsfrom DO11.10 OVA-specific TCR-transgenic mice to a TH2 or TH17 phenotype by culturing in conditioned medium. In addition,we also tested the steroid responsiveness of TH2 and TH17 cells. In vitro, TH17 cytokine responses were not sensitive to dexa-methasone (DEX) treatment despite immunocytochemistry confirming glucocorticoid receptor translocation to the nucleus fol-lowing treatment. Transfer of TH2 cells to mice challenged with OVA protein resulted in lymphocyte and eosinophil emigrationinto the lung that was markedly reduced by DEX treatment, whereas TH17 transfer resulted in increased CXC chemokinesecretion and neutrophil influx that was not attenuated by DEX. Transfer of TH17 or TH2 cells was sufficient to induce airwayhyperresponsiveness (AHR) to methacholine. Interestingly, AHR was not attenuated by DEX in the TH17 group. These datademonstrate that polarized Ag-specific T cells result in specific lung pathologies. Both TH2 and TH17 cells are able to induce AHR,whereas TH17 cell-mediated airway inflammation and AHR are steroid resistant, indicating a potential role for TH17 cells insteroid-resistant asthma. The Journal of Immunology, 2008, 181: 4089–4097.

T he CD4� Th cells are classified as TH1, TH2, and TH17based on their cytokine expression profile. Classically, re-cruitment of TH2 lymphocytes that secrete IL-4, IL-5,

IL-9, and IL-13 accompanied by eosinophil recruitment to the air-ways has been considered integral to the pathogenesis of asthma.Inflammatory cell recruitment to the lung results in tissue damage,mucus hypersecretion, bronchoconstriction, and airway hyperre-sponsiveness (AHR).3 Glucocorticoid use attenuates airway eosin-ophilia by inducing eosinophil apoptosis and inhibiting the re-sponse to IL-5 and GM-CSF survival signals (1, 2). The broaddistribution of the glucocorticoid receptor in the airways suggeststhere are multiple cell targets including not only inflammatorycells, such as mast cells and the aforementioned eosinophil, butepithelial and airway smooth muscle cells (3). Studies have shownthat 50% of asthma cases are noneosinophilic and in these cases

CXCL8-mediated neutrophil inflammation in the airways is pre-dominant (4).

TH17 cells are a distinct population of CD4� T cells that secreteIL-17A, IL-17F, and IL-22 (5, 6). The importance of TH17 cells inneutrophilic inflammation lies in the ability of IL-17 to inducegranulopoiesis, neutrophil chemotaxis, and the antiapoptotic prop-erties of G-CSF (7, 8). IL-17 is increased in the sputum of asth-matic patients and correlates with CXCL8 levels and the numberof neutrophils in the sputum. PBMCs from both atopic and nona-topic asthmatics secrete IL-17 in response to anti-CD3 and anti-CD28 Ab stimulation (9). Apoptosis of neutrophils is inhibited byglucocorticoids (10) and numerous studies have suggested non-eosinophilic asthma is associated with poor response to cortico-steroid treatment (11, 12). Based on this, we hypothesized thatTH17 cells mediate airway inflammation and hyperresponsivenessassociated with noneosinophilic asthma and are not responsive toglucocorticoid treatment.

Models using adjuvant-induced Ag sensitization with alum haveshown that IL-17A and IL-17RA KO mice have reduced delayed-type hypersensitivity reactions in tissue (13) and reduced primingof TH2-immune responses in lung lymph nodes (Ref. 14 and ourunpublished observations). This may be due to the recently de-scribed role of IL-17 in germinal center formation and setting upchemokine gradients in lymph nodes (15). Thus, upon Ag chal-lenge, IL-17RA KO mice have shown reduced recruitment of bothneutrophils and eosinophils into the airway (14). Whether this isdue to a role for IL-17RA in T cell priming in the lymph nodes orin the effector phase after Ag challenge remains unclear (14).Moreover it remains unclear from the studies published to datewhether TH17 cells in and of themselves mediate airway hyperre-sponsiveness and goblet cell hyperplasia independent of Th1 orTH2 cells. Based on these data, we used a model that had previ-ously been used to study the effect of TH2 cells in inducing airwayinflammation and airway hyperresponsiveness utilizing adoptive

*Department of Pediatrics, Lung Immunology and Host Defense Laboratory and †De-partment of Medicine, Division of Pulmonary, Allergy, and Critical Care Medicine,University of Pittsburgh, Pittsburgh, PA 15213; and ‡Department of Medicine, Ver-mont Lung Center, University of Vermont, Burlington, VT 05405

Received for publication May 7, 2008. Accepted for publication July 20, 2008.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by National Heart, Ling, and Blood Institute GrantR01HL079142 and National Institute of Allergy and Infectious Disease Grant5T32AI060525.2 Address correspondence and reprint requests to Dr. Jay K. Kolls, Children’s Hos-pital of Pittsburgh, Suite 3765, 3705 Fifth Avenue, Pittsburgh, PA 15213. E-mailaddress: jay.kolls@chp.edu3 Abbreviations used in this paper: AHR, airway hyperresponsiveness; Ad, adenovi-rus; BAL, bronchoalveolar lavage; DEX, dexamethasone; GR, glucocorticoid recep-tor; i.t., intratracheal; LH, lung homogenate; KO, knockout; rm, recombinant mouse;WT, wild type; PMN, polymorphonuclear neutrophil; PAS, periodic acid-Schiff.

Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00

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transfer of polarized T cell populations to SCID mice (16). SCIDmice were chosen to isolate the contribution of the transferred cellsas opposed to contaminating endogenous CD4� T cell popula-tions. Additionally, this model does not use adjuvant priming invivo which has recently been questioned in terms of its relevanceto airway sensitization, which is what occurs in human allergensensitization.

Presently, we report that in vitro-polarized TH17 cells are non-responsive to glucocorticoids. Adoptive transfer of these cells in amodel of Ag-induced airway inflammation resulted in increasedCXC chemokine secretion and G-CSF in the lung that was asso-ciated with neutrophil influx to the airways. Treatment of TH17cell reconstituted mice with dexamethasone (DEX) did not alterairway inflammation but did attenuate TH2-induced airway inflam-mation. Reconstitution of mice with either TH2 or TH17 cells re-sulted in increased AHR to methacholine challenge. Surprisingly,DEX treatment significantly reduced AHR in mice that receivedTH2 cells but not in TH17-reconstituted animals. These data dem-onstrate the significance of TH17 cellular responses in airway dis-ease and implicate these cells as having a role in steroid-resistantasthma.

Materials and MethodsMice

Six- to 8-wk-old, male BALB/c mice and SCID mice were purchased fromThe National Cancer Institute. C.DO11.10 TCR-transgenic mice were ob-tained from The Jackson Laboratory. IL-17R null mice have been previ-ously described (17). All animals were housed in a pathogen-free environ-ment and given food and water ad libitum. All experiments were approvedby the Children’s Hospital of Pittsburgh Animal Research and CareCommittee.

Abs and reagents

Cell culture. Recombinant mouse (rm) IL-2, rmIL-4, rmIL-6, rmIL-23,porcine TGF-�, anti-mouse IL-4 (clone 30340.11), and anti-mouse IFN-�(clone 37895) were purchased from R&D Systems.Immunofluorescence. Rabbit anti-mouse glucocorticoid receptor (GR; Af-finity BioReagents) and goat anti-rabbit IgG conjugated to Alexa Fluor 555(Invitrogen) were used.Western blot. Polyclonal rabbit anti-mouse GR (Affinity BioReagents),mouse monoclonal IgG1 anti-chicken �-actin (Santa Cruz Biotechnology),goat anti-rabbit IgG conjugated to alkaline phosphatase (AP; Bio-Rad), andgoat anti-mouse IgG conjugated to AP (Santa Cruz Biotechnology)were used.

In vitro differentiation of Th cell subsets

CD4� T cells from the spleens and lymph nodes of C.DO11.10 TCR-transgenic mice were enriched by negative selection using magnetic beads(Miltenyi Biotec). CD4�CD62L�CD25� cells were sorted on a FACSAria(BD Biosciences); purity was consistently �99%. Cells were cultured withirradiated APCs pulsed with OVA peptide 323–339 (OVA323–339) underpolarizing conditions as previously reported (18). Briefly, medium wassupplemented with 20 U/ml IL-2, 5 ng/ml rmIL-4, and 10 �g/ml anti-IFN-� (for TH2 polarization), or 10 ng/ml rmIL-23, 1 ng/ml TGF-�, 20ng/ml rmIL-6, 10 �g/ml anti-IL-4, and anti-IFN-� (for TH17 polarization).Cultures were split 1:2 on day 3 and harvested on day 6. The total numberof cells was determined by counting on a hemocytometer; viable cells weredefined by trypan blue exclusion. APCs alone were CD4neg or CD4dim andproliferating CD4� T cells were CD4bright and stained positively withKJ1-26 (eBioscience) and anti-Do11.01 TCR Ab. CD4bright cells were se-lected for immunohistochemistry studies.

In vitro assays

To confirm the phenotype of the Th cell subsets, cells were stimulated within vitro with PMA/ionomycin for ELISPOT assays (R&D Systems) oranti-CD3/anti-CD28-coated microbeads (Dynal) for cytokine assays.Where indicated, cells were pretreated with increasing doses of DEX (SicorPharmaceuticals) for 2 h before the addition of microbeads. After 36 h inculture, cell supernatants were collected and stored at �80° until analysis.To measure NFAT activity of polarized T cells, primary culture mouse Tcells (1 � 106/1.5 ml) were transfected with 4 �g of NFAT-luciferase

plasmid (gift from S. Gaffen, State University of New York, Buffalo, NY)and 1 �g of SV40-Renilla. Transfection was conducted with the Mouse TCell Nucleofector Kit (Amaxa Biosystems) using the X-005 program onthe Nucleofector II instrument (Amaxa Biosystems). Following transfec-tion, T cells were cultured for 18 h and were then treated with or without1 �M DEX for 2 h before treatment with anti-CD3/anti-CD28-coatedDynabeads (Invitrogen Dynal) for 6 h at 37oC. After stimulation, luciferaseactivity was determined using the Dual-luciferase Reporter Assay System(Promega). NFAT-luciferase levels were normalized to SV40-Renilla as aninternal transfection control.

Immunocytochemistry

Superfrost Plus Micro Slides (VWR International) of 100,000 cells CD4�

cells (selected by Miltenyi beads) were fixed in 100% ice-cold methanolfor 10 min. Slides were stained with rabbit anti-mouse GR and visualizedwith goat anti-rabbit IgG conjugated to Alexa Fluor 555 (Invitrogen) aspreviously described (19). Accumulation of GR in the nucleus was ob-served by epifluorescence illumination with a Zeiss Axioplan 2 microscopeequipped with a �63, 1.4 aperture oil immersion objective. Quantitation offluorescence was performed by analyzing regions of interests (drawn overthe 4�,6-diamidino-2-phenylindole nuclear signal) of 100 cells capturedwith a charge-coupled device camera (Intelligent Imaging Innovations) andrelative intensity values were determined and averaged with SlideBooksoftware (Intelligent Imaging Innovations).

Western blot analysis

Nuclear and cytoplasmic protein fractions were extracted from differenti-ated T cells after DEX treatment using the Nuclear Extract Kit (ActiveMotif) according to the manufacturer’s instructions. Protein concentrationwas determined using a BCA Protein Assay Kit (Pierce Biotechnology).AP development was visualized by the AP Conjugate Substrate Kit (Bio-Rad) according to the manufacturer’s instructions.

Adoptive transfer model

BALB/c SCID mice were challenged with OVA protein (50 �g/mouse,intratracheal (i.t.); Sigma-Aldrich) on day �1. On day 0, 1 � 106 differ-entiated Th cells were adoptively transferred by retro-orbital injection.Mice were challenged with OVA for 3 consecutive days after cell transfer(50 �g/mouse/day, i.t.). Animals were sacrificed 24 h after the last airwaychallenge. Where indicated, mice were treated with 2.5 mg/kg DEX or PBScontrol 2 h before cell transfer on day 0 and before OVA challenge on day2. In separate experiments, wild-type (WT) BALB/c mice and IL-17R nullmice on a BALB/c background underwent the same adoptive transfer andOVA challenge protocol.

Bronchoalveolar lavage (BAL) fluid and lung tissue collection

Lungs were lavaged with five 1-ml volumes of calcium- and magnesium-free PBS. The supernatant from the first 1-ml aliquot was stored at �80°Cfor later analysis. Cell pellets from both aliquots were pooled in 1 ml andtotal white blood cells in BAL fluid were counted using a particle counter(Z1; Beckman Coulter). Cytospins of 100,000 cells were prepared using aShandon Cytospin 4 (Thermo Electron), slides were stained with HEMA 3(Fisher Scientific), and differentials were quantified by counting 100 cells.

Lungs were harvested following the collection of lavage fluid. The rightlungs were homogenized in 1 ml of PBS containing 0.05% Triton X-100and Complete Protease Inhibitor (Roche). Homogenate was centrifuged at12,000 � g for 15 min and supernatant was stored at �80°C for latercytokine analysis.

Cytokine analysis

Cell supernatants, BAL fluid, and lung homogenate (LH) samples wereanalyzed for protein levels of G-CSF, IL-4, IL-5, IL-6, IL-13, IL-17,IFN-�, and KC using a Luminex multiplex suspension cytokine array(Linco) according to the manufacturer’s instructions. The data were ana-lyzed using Bio-Plex Manager software (Bio-Rad). IL-22 was measuredusing a commercial ELISA (R&D Systems) following instructions pro-vided by the manufacturer.

Overexpression of IL-4 and IL-17 in the lungs

Adenovirus expressing IL-4, IL-17, or luciferase as a control was given at108 PFU i.t. on day 0 and AHR was determined 72 h later. Overexpressionof IL-4, IL-13, and IL-17 was determined by Luminex in both the BALfluid and LH of recipient mice.

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Determination of AHR

Airway responsiveness to methacholine challenge was determined on day4 as previously described using anesthetized, intubated mice connected toa computer-controlled small-animal mechanical ventilator (flexiVent; SCI-REQ) (20). Airway resistance values (Rn) were calculated in response toprogressive concentrations of methacholine administered by i.v. injection.

Statistical analysis

Data are reported as mean � SEM. GraphPad Prism version 4.0 was usedto calculate p values using one-way ANOVA with a Tukey multiple com-parison posttest. A value of p � 0.05 was considered statisticallysignificant.

ResultsTH17 cells are resistant to DEX treatment in vitro

Studies have shown that glucocorticoids inhibit the production ofIL-4, IL-5, and IL-13 from TH2 cells (21). To test the sensitivity ofTH17 cells to glucocorticoids, naive CD4� T cells from DO11.10TCR-transgenic mice were differentiated in the presence of IL-6,IL-23, TGF-�, anti-IL-4, and anti-IFN-� (18). As a positive con-trol, naive CD4� T cells were polarized toward a TH2 phenotypeby conditioning with IL-2, IL-4, and anti-IFN-�. After 6 days inculture, polarization was confirmed by PMA/ionomycin stimula-tion and ELISPOT analysis (Fig. 1, A and B) and secreted cytokineanalysis (Fig. 1, C–F). Only TH17 cells produced IL-17 (Fig. 1A),whereas in TH2 conditions the precursor frequency of IL-4-pro-ducing T cells was 5.6-fold greater in TH2 cells compared with Tcells grown in TH17 conditions (Fig. 1B). Cells were also har-vested on day 6 and treated with increasing doses of DEX for 2 hbefore stimulation with anti-CD3/anti-CD28-coated microbeads.

As expected, TH2 cells secreted IL-4 (data not shown), IL-5, andIL-13 (Fig. 1, C and D) following CD3/CD28 stimulation andTH17 cells secreted high levels of IL-17 and IL-22 (Fig. 1, Eand F). Although both IL-5 and IL-13 generation from TH2cells were inhibited at all doses of DEX studied, TH17 cellcytokine production was not sensitive to DEX treatment at anydose tested. Apoptosis was analyzed by annexin V and 7-ami-noactinomycin D staining and �85% of the TH2 and TH17 cellswere annexin V and 7-aminoactinomycin negative (viable) after1 �M DEX treatment (data not shown).

DEX treatment inhibits Ag-specific TH2-mediated airwayinflammation but not TH17-mediated airway inflammation in anadoptive transfer model

To determine the Ag-specific immune response elicited by effectorTH17 cells in vivo, an adoptive transfer model of airway Ag-in-duced inflammation was used (Fig. 2A). In vitro-polarized TH cellsubsets were transferred i.v. to SCID mice on a BALB/c back-ground that had been challenged with OVA protein i.t. 1 day be-fore cell transfer to promote cell migration to the airways (16).Following cell transfer, mice were challenged with OVA i.t. for 3consecutive days and sacrificed on day 4, 24 h after the last OVAchallenge. Mice were treated with DEX (2.5 mg/kg) or PBS con-trol by i.p. injection 2h before cell transfer on day 0 and again 2 hbefore OVA challenge on day 2. Control mice reconstituted withTH2 cells that were challenged with OVA had increased levels ofIL-5 and IL-13 in LH (Fig. 2, B and C). Mice transferred TH2 cellswithout OVA challenge or scid mice challenged with OVA with-out T cell transfer had LH levels of IL-5 and IL-13 that were �50

FIGURE 1. TH17 cells are not sen-sitive to DEX treatment in vitro.CD4�CD62L�CD25� naive T cellsisolated from DO11.10 OVA TCR-transgenic mice were cultured with WTBALB/c splenocytes that had beenpulsed with OVA323–339 under polariz-ing conditions. On day 6, cells werecollected and stimulated with PMA/ionomycin for precursor frequency byELISPOT or pretreated for 2 h with theindicated doses of DEX before stimula-tion with CD3/CD28 beads � IL-2. A,Spot frequencies of IL-17-producing Tcells. B, Spot frequencies of IL-4-produc-ing T cells. Cells were plated under thefollowing conditions: T cells indicate un-stimulated T cells cultured without beadstimulation; 1 �M DEX indicates T cellspretreated with 1 �M DEX but not stim-ulated with CD3/CD28 microbeads;beads indicates untreated T cells stimu-lated with CD3/CD28 microbeads; �MDEX � bead indicates T cells pretreatedwith the indicated dose of DEX and stim-ulated with CD3/CD28 microbeads. Cellculture supernatant was collected at 36 hafter stimulation and the levels of TH2and TH17 cytokines were determined bymultiplex suspension array assay. C,IL-5 levels from TH2 cells. D, IL-13levels from TH2 cells. E, IL-17 levelsfrom TH17 cells. F, IL-22 levels fromTH17 cells. All data are graphed asmean � SEM for n � 5–8; �, p � 0.01.

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pg/ml (data not shown). Treatment with DEX significantly inhib-ited the lung levels of IL-5 and IL-13 (Fig. 2, B and C). Groupsreconstituted with TH17 cells showed an increase in IL-17, G-CSF,and the mouse homolog of CXCL8 KC in LH that was not ob-served in mice receiving TH2 cells (Fig. 2, D–F). Mice transferredTH17 cells without OVA challenge or scid mice challenged withOVA without T cell transfer had LH levels of IL-17 and G-CSFthat were �100 pg/ml; KC levels were �500 pg/ml in these con-trol mice (data not shown). Concurrent with the in vitro glucocor-ticoid resistance, DEX treatment did not attenuate the TH17-in-duced inflammatory cytokine response; in fact, there was a trendtoward increased levels of KC in the LH of DEX-treated TH17-reconstituted mice compared with the TH17 PBS control.

Transfer of either TH2 or TH17 cells resulted in specific cellularinflux into the airways. We initially assessed lymphocyte recruit-ment into the lung 24 h after adoptive transfer. Both transferof TH2 cells and TH17 cells resulted in 0.15 � 0.06 � 106 and0.18 � 0.07 � 106 CD3/CD4� cells in BAL ( p � NS). However,after three challenges with OVA, differential counting of BAL

fluid cytospins showed that TH2 reconstitution resulted in airwayinflammation consisting of both eosinophils and lymphocytes andto a lesser extent polymorphonuclear neutrophils (Fig. 3, A–C).Mice transferred TH2 cells without OVA challenge or scid micechallenged with OVA without T cell transfer had BAL eosin-ophil counts that were �0.1 � 106/ml (data not shown). Theinflux of lymphocytes and eosinophils was highly sensitive toDEX (Fig. 3, A and B), whereas the small numbers of polymor-phonuclear neutrophils in the BAL were not inhibited by DEXtreatment (Fig. 3C). As expected, TH17 transfer resulted in aprimarily neutrophilic airway response (Fig. 3C). Mice trans-ferred TH17 cells without OVA challenge or scid mice chal-lenged with OVA without T cell transfer had BAL neutrophilcounts that were �0.2 � 106/ml (data not shown). In contrast tomice receiving TH2 cells, DEX treatment exacerbated airwayneutrophilia in TH17-reconstituted mice.

To determine whether the airway responses of reconstitutedmice were mediated by IL-17 per se or another product of TH17cells, we adoptively transferred TH17 cells into IL-17R KO mice

FIGURE 2. In vivo cytokine and chemokine profiles induced by TH17 cell transfer and allergen provocation are not attenuated by DEX. A, SCID miceon a BALB/c background were challenged with OVA protein (50 �g/mouse) i.t. on day �1. On day 0, mice were pretreated with DEX (2.5 mg/kg, i.p.)or PBS control 2 h before cell transfer (1 � 106 in vitro-polarized TH cells). Mice were subsequently challenged with 50 �g/mouse OVA for 3 consecutivedays. Animals were pretreated with DEX on day 2, 2 h before challenge. Mice were sacrificed on day 4, 24 h after the last airway challenge. Cytokine andchemokine levels in the BAL fluid (data not shown, unless indicated) and LH (B–E) were determined by multiplex suspension cytokine array. Data aregraphed as mean � SEM for n � 4–8; �, p � 0.05 TH2_PBS vs TH2_DEX.

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on a BALB/c background and challenged them with OVA 1 daybefore and for 3 consecutive days after cell transfer as before (Fig.2A). IL-17R KO mice had significantly attenuated KC, G-CSF, andIL-6 responses compared with WT BALB/c controls (Fig. 4, A–C).Consistent with the decrease in CXC chemokine production inIL-17R KO mice, there was a significant decrease in the number ofneutrophils in the airways (Fig. 4D). Again, there was not a sig-nificant increase in eosinophil recruitment to the lung in either WTor IL-17R KO mice transferred TH17 cells and challenged withOVA (Fig. 4E). These data indicate that the inflammatory cytokine

response and cellular influx associated with TH17 cell transfer inthis model system is mediated primarily by IL-17 signalingthrough the IL-17R.

Ag-induced AHR following adoptive transfer of TH17 cells isnot attenuated by DEX treatment

Having established the airway inflammation induced by TH17 celltransfer, we wanted to determine whether TH17 cells are sufficientto induce mucus hyperplasia and AHR at a level comparable to

FIGURE 3. TH2- and TH17-mediated air-way inflammation and AHR. BAL fluid dif-ferential was determined by counting at least100 cells from cytospins prepared of100,000 cells. Data for A, lymphocytes; B,eosinophils; C, neutrophils; and D, macro-phages are graphed as mean � SEM per-centage of total cells for n � 4–6; �, p �0.05 PBS vs DEX in same transfer group; #,p � 0.05 TH2_PBS vs TH17_PBS.

FIGURE 4. The inflammatory response associated with TH17 cell transfer is mediated by IL-17. TH17 cells were transferred to IL-17R KO mice andBALB/c controls that had been challenged with OVA 1 day before transfer. Mice were challenged with 50 �g of OVA per mouse for 3 consecutive daysafter transfer. BAL fluid differential and LH levels of cytokines and chemokines 24 h after the last airway challenge were determined. Levels of KC (A),G-CSF (B), and IL-6 (C) in the LH were determined by multiplex suspension cytokine array. BAL fluid neutrophils (D), and eosinophils (E) are expressedas cells/ml � 106. Data are graphed as mean � SEM for n � 4–6; �, p � 0.05 compared with WT control.

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that observed following transfer of TH2 cells. A significant hall-mark of allergic airway disease is mucus hyperplasia; to addressthis, LH expression of gob5 was determined by real-time PCR.Data are normalized to control mice that had been challenged withOVA on days �1, 1, 2, and 3 but received a mock PBS transfer onday 0. TH2 transfer resulted in a marked significant increase ingob5 gene expression in the lung (Fig. 5A), which was decreasedin the DEX treatment group (Fig. 5B). Expression of gob5 in TH17cell transfer groups was also significantly elevated over mock-transferred animals (200-fold increase compared with OVA-chal-lenged mice without cell transfer) but was 10-fold lower thanthat of the TH2 transfer groups (Fig. 5A). However, compared withthe TH2 transfer groups, DEX treatment had no effect on gob5

gene expression in the TH17 cell transfer group. Expression ofgob5 was confirmed by periodic acid-Schiff (PAS) staining of lungsections; Fig. 5B is representative sections from TH2 and TH17 cellrecipient animals treated with PBS or DEX, respectively. BothTH2 and TH17 transfer groups exhibit positive PAS staining inairway epithelial cells.

To date, there are no data to demonstrate whether TH17 cellsare sufficient to induce AHR. IL-17 levels in the sputum cor-relate with AHR to methacholine in asthmatic and chronic bron-chitis patients (11). To determine whether IL-17 was sufficientto induce AHR, an adenovirus (Ad) overexpressing IL-17(AdIL-17) was administered i.t. to WT BALB/c mice and AHRto methacholine was measured 72 h later. As controls, separate

FIGURE 5. IL-17 is sufficient to induce mucus secretion and AHR. A, gob5 expression was determined in whole lung by quantitative real-time PCR andnormalized to the housekeeping gene 18S. Data are graphed as mean � SEM for n � 4; �, p � 0.05 vs TH2_PBS. B, Expression data were confirmed byPAS staining of lung sections from TH2, and TH17, transferred animals. C, An adenovirus overexpressing IL-4, IL-17, or luciferase control was given i.t.to WT BALB/c (108 PFU/mouse) and airway responsiveness to methacholine was determine on day 3. �, p � 0.05 AdIL-4 vs Adluc; �, p � 0.5 AdIL-17vs Adluc. D, Methacholine dose-response curves in control, TH2 vehicle-treated, or TH2 DEX-treated mice. Control mice received all OVA challenges butPBS i.v. at time of cell transfer to experimental groups. Data are graphed as mean � SEM for n � 6–8; �, p � 0.05 for TH2 vs TH2 � DEX. E,Methacholine dose-response curves in control, TH17 vehicle- treated, or TH17 � DEX-treated mice. Control mice received all OVA challenges but PBSi.v. at time of cell transfer to experimental groups. Data are graphed as mean � SEM for n � 6–8.

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groups of mice were given an adenovirus overexpressing lucif-erase (Adluc) or IL-4 (AdIL-4) which induces local IL-13 andincrease in AHR in response to methacholine (mean IL-13 con-centrations in AdIL-4-treated mice was 358 � 68 pg/ml vs5.6 � 2.2 in Adluc-treated mice). Overexpression of IL-4 orIL-17 in the lung was sufficient to cause significant increases inairway resistance in response to methacholine challenge (Fig.5C). AdIL-4 caused a greater shift in the methacholine dose-response curve with significantly higher airway resistance (Rn)values in mice administered the mid-range dose of 12.5 mg/ml.At the 6.25-mg/ml dose and the 25-mg/ml dose, there was nodifference between AdIL-4 and AdIL-17, but both were signif-icantly higher than Adluc control mice. These data that showIL-17 overexpression is sufficient to induce AHR to methacho-line in WT mice.

We next examined the AHR mediated by TH2 and TH17 cellsin the adoptive transfer model of Ag-specific airway inflamma-tion (Fig. 2A). AHR to methacholine was determined 24 h afterthe last airway challenge. Fig. 5D depicts the dose response ofmethacholine in control mice, mice that received TH2 cellstreated with vehicle, or mice transferred TH2 cells treated withDEX. Ag-challenged mice that received TH2 cells and vehicletreatment showed a significant leftward shift of the methacho-line dose-response curve compared with Ag- challenged controlmice that did not receive T cells (Fig. 5D). This shift was sig-nificantly attenuated with DEX treatment (Fig. 5D). Ag-chal-lenged mice that received TH17 cells and vehicle treatment alsoshowed a significant leftward shift of the methacholine dose-response curve compared with Ag-challenged control mice thatdid not receive T cells (Fig. 5E). However, in contrast to TH2cell-transferred mice, this shift was significantly attenuatedwith DEX treatment (Fig. 5E).

Mechanism of enhanced TH17 cellular responses to DEXtreatment

Glucocorticoids exert their effects through binding the GR, result-ing in nuclear translocation and transactivation of genes containingglucocorticoid-response elements (12). To determine whetherTH17 cells showed a defect in GR� binding to DEX resulting indiminished translocation to the nucleus, GR� localization was vi-sualized by immunocytochemistry in in vitro-polarized TH cellsubsets (Fig. 6A). Fluorescent staining showed that the GR� isprimarily cytoplasmic before treatment with DEX, and DEX treat-ment resulted in a significant increase in nuclear GR� in both celltypes. This observation was confirmed by Western blot, whichshowed a decrease in cytoplasmic GR� content (Fig. 6B). Fluo-rescent microscopy analysis showed a significant increase in nu-clear GR� in both TH2 and TH17 cells 30 min after DEX treatment(Fig. 6C). These results indicate that TH17 cells do express GR�and are not deficient in their ability to translocate GR� to thenucleus upon glucocorticoid treatment.

Although the ability of GR� to bind glucocorticoids and trans-locate to the nucleus is intact, the defect may lie at the level ofDNA binding. Suppression of IL-5 by glucocorticoids involvesrepression of GATA-3 signaling mediated by GR� binding to theIL-5 NFAT/AP-1-response element and subsequent recruitment ofhistone deacetylase (22). Studies have shown that IL-17 expressionin mouse and human CD4� T cells is dependent on the NFAT andMAPK pathways (23, 24). Activated GR� may be unable to re-press NFAT binding to the IL-17 locus, which would explain thefact that IL-17 production is not attenuated by DEX. Althoughthere are several mechanisms by which TH17 cells might preventactivated GR� from repressing NFAT activity, up-regulation ofNFAT expression in response to DEX is one way by which the cellcould mask the drug’s effects. To test this hypothesis, polarized

FIGURE 6. Mechanism of TH17 cell resistance to DEX treatment. TH17 cells are not deficient in their ability to translocate GR�. A, GR staining byimmunofluorescent microscopy in DEX-treated (1 �M) and nontreated TH2 and TH17 cells. B, Cytoplasmic protein fractions of DEX-treated and nontreatedTH2 and TH17 cells were immunoblotted for GR� and �-actin. Samples were loaded in the following order: TH2_PBS, TH2_DEX, TH17_PBS, andTH17_DEX. C, Florescent intensity of nuclear GR� staining in TH2 and TH17 cells with and without DEX treatment. Data are graphed as mean � SEM;�, p � 0.05 for DEX compared with no DEX conditions. D and E, TH2 (C) or TH17 (D) cells were transfected with NFAT-luciferase before stimulationwith anti-CD3 and anti-CD28 microbeads (6 h) with or without pretreatment (2 h before beads) with DEX; �, p � 0.05 control (CNTRL) vs CD3/CD28;#, p � 0.05 CD3/CD28 vs CD3/CD28 � DEX.

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TH2 and TH17 cells were transfected with a NFAT luciferase re-porter plasmid and were stimulated with anti-CD3 and anti-CD28microbeads in the presence or absence of DEX (Fig. 6, C and D).DEX significantly inhibited NFAT luciferase activity in TH17cells, discounting the concept that NFAT activity is steroid inde-pendent in TH17 cells.

DiscussionWe have shown that reconstitution of SCID mice with TH2 orTH17 cells in a model of Ag-induced airway inflammation lead todifferential chemokine profiles and cellular influx to the airways.Adoptive transfer of TH17 cells resulted in increased levels ofCXC chemokines and G-CSF in the BAL fluid and LH followingAg provocation. We do not believe this is due to homeostatic pro-liferation due to the short time frame of the transfer model (25) aswell as similar findings were observed in WT mice (Fig. 4). More-over, the experiment in WT mice vs IL-17RA KO mice demon-strated that IL-17RA signaling is required for the induction of KCand G-CSF, as well as neutrophil recruitment after TH17 T celltransfer (Fig. 4). This was associated with neutrophil influx to theairways and is exacerbated by DEX treatment. Animal-transferredTH2 cells in this model exhibit a typical TH2 phenotype charac-terized by the influx of lymphocytes and eosinophils to the airwaysand are sensitive to DEX treatment. The chemokine and inflam-matory responses elicited by TH17 cell transfer primarily involvessignaling through the IL-17R. This is consistent with the recentstudy by Liang et al. (26) that showed that IL-22, another productof TH17 cells, played a limited role in neutrophil recruitment in anadoptive transfer model compared with IL-17A or IL-17A/F het-erodimer. Despite differential chemokine and cellular responses,both TH2 and TH17 recipient animals exhibited increased AHRand mucus secretion. TH17 cells secrete IL-17A and F, IL-6, IL-22, and TNF-� (5). Transfer of TH17 cells to IL-17R KO miceshowed a substantial decrease in CXC chemokine expression, G-CSF, IL-6 and lung neutrophilia confirming that the TH17 cells aremigrating to the airways to initiate a response and IL-17R signal-ing is necessary for the phenotype associated with their transfer.However, the individual contributions of IL-17A or IL-17F ho-modimers and IL-17A/F heterodimers in AHR remains to bedetermined.

It has been suggested that IL-17 modulates allergic airway in-flammation by dampening Th2 cytokine responses (14, 27). Onestudy showed that Ab neutralization of IL-17 before OVA chal-lenge in sensitized mice resulted in decreased neutrophil influx tothe airways but increased airway eosinophilia that those authorsattributed to enhanced serum and BAL fluid IL-5 levels in theseanimals (27). We found that the transfer of TH17 cells to WTBALB/c mice resulted in very low and variable Th2 cytokine re-sponses and the IL-17R KO mice did not exhibit an exacerbatedTh2 response. One explanation of these divergent findings is thatIL-17 signaling is required for the initiation of Ag-mediated air-way inflammation but functions to dampen Th2 responses there-after. This was suggested by studies showing that IL-17R KO miceexhibited decreased airway eosinophilia and IL-5 in an OVA-in-duced model of pulmonary inflammation, while treating WT ani-mals with exogenous IL-17 reduced Th2 cytokine levels in BALand LH and eosinophil influx (14). The former IL-17R KO data arein agreement with what we have found in our model of TH17-induced airway inflammation. To date, the ability of TH17 cells todirectly regulate Th2 cells has not been shown. However, regula-tion between Th2 and TH17 may not be as important as the pos-sibility that these Th cell populations are mediating distinct endo-phenotypes of asthma. Asthma can be classified as atopic (allergic)and nonallergic. Patients with nonatopic asthma do not have ele-

vated IgEs, tend to have more airway neutrophilia, and are clini-cally steroid resistant (12). The data presented in our model systemsupport a role for TH17 cells in nonatopic disease.

DEX treatment of TH17 cell-reconstituted animals resulted inincreased numbers of neutrophils in the airways following allergenchallenge. The mechanism of increased neutrophil numbers inDEX-treated animals reconstituted with TH17 cells is not clear.The expression of IL-17 and CXCL8 is increased in the sputum ofasthmatic patients and positively correlates with each other andwith neutrophil levels in the sputum (28). Our data suggest thatincreased levels of neutrophil chemoattractants and G-CSF, whichhas been shown to be an important survival factor for neutrophils,may be a mechanism of enhanced recruitment and survival of neu-trophils following DEX treatment in TH17-reconstituted animals.This is fitting with published reports that corticosteroids down-regulate the CC chemokines CCL2 and CCL11 while exacerbatingthe CXCL8 response in the airways of asthmatic patients (29). Thissame study found that corticosteroids decreased airway eosino-philia but increased the number of neutrophils in the airways. Neu-trophils are relatively steroid resistant compared with T cells andeosinophils and glucocorticoids enhance survival of neutrophils invitro (10, 30). In vitro neutralization of another neutrophil survivalfactor GM-CSF did not alter neutrophil survival following glu-cocorticoid treatment (31). Glucocorticoids exert their effectsthrough binding the GR, resulting in nuclear translocation andtransactivation of genes containing glucocorticoid-response ele-ments (32). It has been suggested that human neutrophils expresshigher levels of the dominant-negative isoform of the GR, GR�(19), rendering them nonresponsive to glucocorticoids. However,murine cells do not express GR� and we observed equivalent nu-clear translocation of GR to the nucleus of Th2 and TH17 cells anda similar ability to inhibit NFAT transcriptional activation.

Multiple mechanisms of steroid-resistant asthma have been de-scribed. One study found that steroid-resistant asthmatics fell intotwo categories based on GR-binding and expression patterns; onegroup of patients exhibited increased GR number but reduced GRbinding affinity for glucocorticoids, whereas another subset of ste-roid-resistant patients had a GR-binding affinity similar to that ofsteroid-sensitive patients, but reduced GR expression per cell (33).Future studies will need to examine GR� binding to regions withinthe Il17 and Il22 loci.

In contrast to the TH17 transfer model, in the Th2 transfer modelboth lymphocyte and eosinophil recruitment to the airways of Th2recipient animals treated with DEX and AHR was reduced byDEX treatment. The reduction in lymphocytes may be due to dif-ferential DEX sensitivity of chemokines necessary for T cell re-cruitment or proliferation in vivo. We did not observe differencesin T cell apoptosis of Th2 or TH17 cells in vitro. Both cells showed�85% viability after DEX treatment for 24 h (data not shown).However, this does not exclude a potential role for differentialproliferation or apoptosis in vivo. AHR was significantly improvedwith DEX treatment, but not to the same levels as the effect ofDEX on airway inflammation. AHR is a hallmark feature ofasthma and can generally be separated into two categories, variableAHR that occurs during an allergen-induced late asthmatic re-sponse and persistent AHR. Although some studies link airwayinflammation with AHR, it has been suggested that AHR, espe-cially persistent AHR, can occur in the absence of airway inflam-mation (34). Tournoy et al. (35) showed AHR occurred indepen-dent of eosinophil influx to the airways in a model of house dustmite-mediated airway inflammation. Clinically, administration of amAb to IL-5 to asthmatic patients also significantly decreased thenumber or eosinophils in the sputum without altering AHR. In ourmodel system, AHR is mediated by the CD4� cell populations

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transferred to the mice in a mast cell/IgE-independent manner.Mast cells are resident in vascularized tissue and express highlevels of the IgE receptor Fc�RI. IgE binding and Ag cross-linkingactivates mast cells to release inflammatory mediators includinghistamine, leukotrienes, cytokines. and chemokines, resulting inbronchoconstriction (37). Mast cells can potentiate Th2-mediatedallergic inflammation through the release of IL-5 that is importantin eosinophil survival and the release of histamine that directsdendritic cells to secrete IL-4- polarizing naive cells toward a Th2phenotype (38, 39). Mast cells are sensitive to glucocorticoids;therefore, it is possible that a mast cell-dependent model systemwould show that Th2-induced AHR is more responsive to DEXtreatment.

We have shown that reconstitution of SCID mice with TH17cells in the setting of Ag provocation induces CXC chemokine andG-CSF secretion and neutrophil influx to the airways and is suf-ficient to induce mucus hyperplasia and AHR. In the setting ofTH17 cell transfer, chemokine secretion, cellular influx to the air-ways, and AHR were not sensitive to DEX treatment in thesestudies. Conversely, in the setting of Th2 cell transfer, cytokinesecretion, eosinophil influx to the airways, and AHR were sensi-tive to DEX. These data highlight the complex relationships be-tween airway inflammation and AHR and support the concept thatTH17 cells may be critical mediators of steroid-resistant airwaysinflammation and AHR.

DisclosuresThe authors have no financial conflict of interest.

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