Expression of eotaxin by human lung epithelial cells: induction by cytokines and inhibition by glucocorticoids

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Expression of eotaxin by human lung epithelial cells: induction by cytokines and inhibition by glucocorticoids
   Eotaxin in Human Airway Epithelial Cells  1767  J. Clin. Invest.© The American Society for Clinical Investigation, Inc.0021-9738/97/04/1767/07$2.00Volume 99, Number 7, April 1997, 1767–1773  Expression of Eotaxin by Human Lung Epithelial Cells  Induction by Cytokines and Inhibition by Glucocorticoids   Craig M. Lilly,* Hidetoshi Nakamura,* Howard Kesselman,   ‡   Cathryn Nagler-Anderson,   §   Koichiro Asano,* Eduardo A. Garcia-Zepeda,   ‡   Marc E. Rothenberg,   ‡   Jeffery M. Drazen,* and Andrew D. Luster   ‡   *   Combined Program in Pulmonary and Critical Care Medicine, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115; ‡    Infectious Disease Unit, and §   Mucosal Immunology Laboratory, Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts 02129   Abstract   Eotaxin is a potent and specific eosinophil chemoattractantthat is mobilized in the respiratory epithelium after allergicstimulation. Pulmonary levels of eotaxin mRNA are knownto increase after allergen exposure in sensitized animals. Inthis study we demonstrate that TNF      and IL-1      induce theaccumulation of eotaxin mRNA in the pulmonary epithelialcell lines A549 and BEAS 2B in a dose-dependent manner.Cytokine-induced A549 cell mRNA accumulation was max-imal at 4 h and was significantly enhanced when the cellswere costimulated with IFN      . TNF      - and IL-1      –induced in-creases in eotaxin mRNA were diminished in a dose-depen-dent manner by the glucocorticoid dexamethasone and wereaugmented by the protein synthesis inhibitor cycloheximide.Cytokine-induced increases in eotaxin mRNA expression cor-related with increased eotaxin protein production and se-cretion, and dexamethasone inhibition of cytokine-inducedeotaxin mRNA augmentation was associated with dimin-ished eotaxin protein secretion. These findings, togetherwith the known kinetics of TNF      and IL-1      mobilization inasthmatic airways and the potent eosinophil chemotactic ef-fects of eotaxin, define a mechanism linking inflammatorycytokine mobilization to eosinophil recruitment that may berelevant to the pathogenesis of asthma. (   J. Clin. Invest.   1997. 99:1767–1773.) Key words: chemokine • eosinophil •   cytokine • asthma • allergic disease   Introduction  The role of inflammation in asthma and other allergic diseasesof the airways is widely appreciated, and airway inflammationis now included as a defining feature of asthma (1, 2). The im-portance of the presence of eosinophils in the airways of pa-tients with fatal asthma has long been recognized (3), but themechanisms by which these cells are recruited and retained inthe lung are only now being elucidated. The importance of air-way eosinophilia in less severe forms of asthma has been docu-mented by studies demonstrating eosinophils in the airways of patients with mild asthma (4–6). These findings and the resultsof other studies linking eosinophil-derived granular proteins(3), arginine-rich proteins, metalloendopeptidases, and lipidmetabolites to airway hyperresponsiveness support the abilityof appropriately activated eosinophils to contribute to asthmasymptoms. The conclusion that tissue eosinophilia is an impor-tant factor in allergic airway disease is based not only on therecognition that eosinophils are present in allergic airways butalso on the correlation of the remission of asthma symptomswith the resolution of airway eosinophilia (7–10). These obser-vations suggest that eosinophils may be necessary to the patho-genesis of asthma and make it likely that the mechanisms bywhich eosinophils are recruited to the airways are relevant toasthma.An increasing but incomplete body of knowledge definesthe mechanisms of eosinophil recruitment, retention, and acti-vation in the airways. Eosinophils respond to a growing list of chemoattractants, including complement factor C5a; platelet-activating factor (PAF); leukotriene B   4  ; IL-2, -3, -5, and -16;and the chemokines IL-8, macrophage inflammatory protein(MIP)   1  -1    , monocyte chemoattractant protein (MCP)-2, MCP-3,MCP-4, RANTES, and eotaxin (11). The importance of cyto-kine mobilization in promoting in vivo airway eosinophil re-cruitment is demonstrated by the effectiveness of IL-5 antago-nists in blocking airway eosinophilia after allergen exposure(12). In addition, IL-5–deficient mice do not develop airwayeosinophilia, lung damage, or airway hyperreactivity, all of which follow aeroallergen challenge of normal mice (13). Therelevance of eosinophil recruitment to asthma is further sug-gested by studies in which IL-5–neutralizing monoclonal anti-bodies administered during the period of sensitization preventthe development of airway eosinophilia and hyperreactivity inrodents and primates (14).Eotaxin is a CC (double cysteine) chemokine that was src-inally isolated as the predominant eosinophil chemoattractantin the lung lavage fluid of sensitized guinea pigs after allergicexposure (15). Eotaxin is unique among eosinophil-activechemoattractants in that it specifically attracts eosinophils.This specificity has recently been demonstrated in vitro forboth mouse (16–18) and human (19, 20) eotaxin in chemotaxisand calcium flux assays. The in vivo potency and specificity of eotaxin have been confirmed in studies demonstrating tissueeosinophil recruitment following instillation of this chemokine  Address reprint requests to Craig M. Lilly, Combined Program inPulmonary and Critical Care Medicine, Department of Medicine,Brigham and Women’s Hospital, 75 Francis St., Boston, MA 02115.Phone: 617-278-0714; FAX: 617-232-4623; E-mail: or Andrew D. Luster, Phone: 617-726-5710; FAX: 617-726-5411; E-mail:  Received for publication 10 April 1996 and accepted in revised form 15 January 1997.  1.  Abbreviations used in this paper:  GAPDH, glyceraldehyde-3-phos-phate dehydrogenase; MCP, monocyte chemoattractant protein;MIP, macrophage inflammatory protein.   Downloaded from on May 15, 2015.   1768  Lilly et al.  into the airways of rodents (17, 18, 21), and following its injec-tion into the skin of rodents (15, 17) and rhesus monkeys (20).Eotaxin is thought to be important in recruiting eosinophils tothe airway after allergic stimulation because the time course of its appearance is congruent with that of eosinophil recruitment(15, 18, 21, 22). Although eotaxin can be produced by severalcell types, production in airway epithelial cells may be particu-larly relevant to allergic diseases, as eotaxin immunoreactivityis detectable in the epithelial cells of nasal polyps that are infil-trated with eosinophils (20). It is increasingly clear that the air-way epithelium participates in the inflammatory process by al-tering its phenotype to produce a variety of mediators andcytokines, including prostaglandins (23), platelet-activatingfactor (24), granulocyte-macrophage colony-stimulating fac-tor (GM-CSF) (25), IL-6 (26), IL-8 (27), MCP-1 (28), andRANTES (29). On the basis of these observations and theknown upregulation of eotaxin after allergic pulmonary stimu-lation, we sought to define the allergic mediators that modu-late the expression of eotaxin mRNA, eotaxin protein produc-tion, and eotaxin secretion in pulmonary epithelial cells.   Methods  Cell culture  A549 cells, derived from a lung adenocarcinoma with the alveolartype II cell phenotype, were obtained from American Type CultureCollection (Rockville, MD). The cells were cultured in F12K mediumwith 10% heat-inactivated FBS. 24 h before stimulation with cyto-kines, this medium was exchanged for an identical formulation notcontaining FBS. BEAS 2B cells, a human bronchial epithelial cell linetransformed by hybrid adenovirus SV-40, were cultured in DMEM-F12 with 10% FBS. Epithelial cells grown to confluence were stimu-lated with geometrically increasing doses of IL-1    , TNF    , and IFN    ,or with 10 nM phorbol PMA, TGF    , 10 ng/ml, or TGF    (5 ng/ml). Inexperiments involving dexamethasone or cycloheximide, the agentswere added 30 min before cell stimulation. In time course experi-ments, the cells were harvested 1, 2, 4, 8, 16, 24, 32, and 48 h afterstimulation. In concentration-response studies, the cells were har-vested at the time of peak expression, which was 4 h after stimulationwith IL-1    or TNF    and 2 h after stimulation with IFN    . Protein syn-thesis was inhibited by the addition of cycloheximide (10   g/ml) tothe medium. This concentration of cycloheximide inhibited [  35  S]me-thionine incorporation into trichloroacetic acid-precipitable proteinby   90%.  RNA analysis  Total RNA was isolated from freshly harvested cells by guanidinium-thiocyanate-phenol chloroform extraction (Stratagene Inc., La Jolla,CA). For Northern analysis, 10–20   g of total RNA was subjected togel electrophoresis on a formaldehyde-2% agarose gel and trans-ferred to a nylon membrane (Schleicher & Schuell Inc., Keene, NH).After UV cross-linking, the membrane was hybridized at 42    C in a50% formamide buffer (pH 7.5), containing 10% dextran sulfate, 5    SSC, 1    Denhardt’s solution, 1% (wt/vol) SDS, 100   g of herringsperm DNA/ml, and 20 mM Tris either with a 32  P-labeled 1.1-kb HindIIIfragment of the human eotaxin gene containing exon 2 and the cod-ing portion of exon 3—with a PCR-generated fragment whose se-quence was confirmed to be identical to that reported for humanRANTES cDNA—or with a glyceraldehyde-3-phosphate dehydroge-nase (GAPDH) cDNA probe (Clontech, Palo Alto, CA). The mem-branes were washed for 40 min at 42    C in 2    SSC–0.1% SDS andthen for 40 min at 55    C in 0.2    SSC–0.1% SDS. To control for RNAloading, the hybridization signal obtained for eotaxin was normalizedto that of GAPDH for each sample. RNA expression was determinedby densitometry as previously reported (22). All experiments involv-ing the quantitation of RNA were performed at least in triplicate anda representative blot is displayed.  Detection of eotaxin protein  Generation of antieotaxin antibodies.  BALB/c mice (Taconic Farms,Germantown, NY) and New Zealand White rabbits (Pocono RabbitFarm and Laboratory, Canadensis, PA) were immunized with recom-binant human eotaxin protein (PeproTech, Rocky Hill, NJ). Spleencells from immunized mice were fused to the P3X63 hybridoma cellline, and monoclonal antibodies were prepared according to standardtechniques. Immune rabbit serum was purified by protein A–Seph-arose (Pharmacia, Piscataway, NJ) affinity chromatography accordingto the manufacturer’s specifications. Immunoblot or ELISA analysisof the antieotaxin monoclonal antibody 2A12 and polyclonal antise-rum demonstrated that these antibodies were specific for human eo-taxin. These antibodies reacted strongly to 100 ng of human eotaxinbut did not react to 100 ng of human MCP-1, 2, 3, 4, MIP-1    , MIP-1    ,or RANTES. Figure 1. Eotaxin mRNA expression in cytokine treated airway epi-thelial cells. (  A ) Northern blot analysis of 20  g total RNA isolated from untreated A549 cells or 4 h after stimulation with the indicated cytokines. Molecular size markers appear on the left of each blot. The blots were hybridizied sequentially with eotaxin-, RANTES-, and GAPDH-specific gene probes. ( B ) Northern blot analysis of 10  g to-tal RNA isolated from untreated BEAS 2B cells 6 or 18 h after stimu-lation with 10 ng/ml TNF  , 5 ng/ml IL-1  , or both. The blots were hy-bridized sequentially with eotaxin, and GAPDH-specific gene probes. Downloaded from on May 15, 2015.   Eotaxin in Human Airway Epithelial Cells  1769  ELISA.  Each well of a high-binding efficiency 96-well ELISAplate was coated with 100 ng of a mouse antieotaxin monoclonal anti-body, designated 2A12. The plate was blocked with a 3% solution of BSA (Sigma Chemical Co., St. Louis, MO) in PBS with 0.02% so-dium azide. After washing with PBS, standards or sample were added;the plate was incubated 2 h at room temperature in a humid environ-ment and was washed again with PBS, and 1   g of a protein A-purifiedfraction of a rabbit antieotaxin polyclonal serum was added to eachwell. The plates were washed twice with PBS after a 2-h room tem-perature incubation; 50   l of a horseradish peroxidase-linked anti–rabbit IgG derived from goats (Kirkegaard & Perry Laboratories,Inc., Gaithersburg, MD) was diluted 1:1000 in blocking buffer andadded to each well. After a 90-min room-temperature incubation, theplates were developed by the 3, 3    , 5, 5    -tetramethylbenzidine mi-crowell-peroxidase substrate method according to the instructions of the manufacturer (Kirkegaard & Perry Laboratories, Inc.). Underthese conditions this assay was sensitive to 50 pg/ml. Cell supernatantwas harvested from 10  7  cells cultured in the absence of stimulation or2, 4, 8, 24, or 48 h after stimulation with IL-1    ; the amount of eotaxinrecovered was calculated from the ELISA concentration and volumeof supernatant and expressed as the amount recovered per 10  6  cells.Cells corresponding to the supernatant samples described above werelysed into 0.5 ml NP-40 lysis buffer, and the eotaxin concentrationwas determined by ELISA and reported as the amount recovered per10  6  cells.   Results  Cytokine-induced eotaxin mRNA expression  Eotaxin mRNA expression was not easily detected by North-ern analysis in A549 or in BEAS 2B cells in the absence of cytokine stimulation. However, stimulation of the cells with0.1 ng of IL-1    /ml or 10 ng of TNF    /ml for 4 h induced the ac-cumulation of a distinct eotaxin mRNA species of   800 bp(Fig. 1  A  ). The addition of 10 nM PMA to the medium was as-sociated with a faint but detectable eotaxin mRNA signal.However, eotaxin mRNA was not detected after 4 h of stimu-lation with IFN    (10 ng/ml), TGF    (10 ng/ml), or TGF    (5 ng/ml). Similarly, RANTES expression in A549 cells was detect-able 4 h after cytokine stimulation with 0.1 ng/ml IL-1    and af-ter 10 ng/ml TNF    (Fig. 1  A  ). Eotaxin mRNA expression inBEAS 2B cells was detectable after stimulation with 5 ng of IL-1    /ml or 10 ng of TNF    /ml with a time course similar tothat of A549 cells. In BEAS 2B cells a synergystic effect of these cytokines was observed (Fig. 1 B  ).  Time course  Treatment of A549 cells with 0.1 ng of IL-1    /ml or 10 ng of TNF    /ml induced maximal eotaxin mRNA expression at 4 h;this activity declined over the subsequent 44 h (Fig. 2,  A  and  B  ). When 100 ng of IFN    /ml was added to the medium, eo-taxin mRNA expression was detected as a faint band appear-ing 2 h after stimulation (Fig. 2 C   ). This is in contrast toRANTES which was maximally induced by IL-1    24 h afterstimulation while maximal expression after TNF-    occurred48 h after stimulation (Fig. 1  A  ; data not shown).  Concentration response characteristic  The addition of geometrically increasing doses of IL-1    to themedium 4 h before harvesting was associated with increasingeotaxin mRNA expression. This effect was maximal at a doseof 1 ng/ml (Fig. 3  A  ). Likewise, the addition of increasing con- Figure 2. Time course of cytokine-induced eotaxin mRNA accumula-tion. Northern analysis of 20  g of total RNA harvested from A549 cells at various times after treatment with 0.1 ng/ml IL-1   (  A ), 10 ng/ml TNF   ( B ), or 100 ng/ml IFN   ( C  ). A quantitative comparison at each time point between the eotaxin-specific signal and the GAPDH-specific signal is illustrated below  A  and B.Figure 3. Dose-response of IL-1  – and TNF  -induced eotaxin mRNA accumulation. Northern analysis of 20  g of total RNA har-vested from A549 cells 4 h after stimulation with increasing concen-trations of IL-1   (  A ) and TNF   ( B ). Below each blot a quantitative comparison is made at each dose between the eotaxin-specific signal and the GAPDH-specific signal. Downloaded from on May 15, 2015.   1770  Lilly et al.  centrations of TNF    to the medium up to a dose of 100 ng/mlwas associated with increasing eotaxin mRNA accumulation(Fig. 3 B  ). Doses of IFN    below 100 ng/ml produced no signaldetectable by Northern analysis (data not shown).  Effects of IFN      The addition of varying concentrations of IFN    to A549 cellsstimulated with 0.1 ng of IL-1    /ml increased the expression of eotaxin mRNA in a dose-dependent manner (Fig. 4  A  ); maxi-mal eotaxin expression was increased twofold over that withIL-1    stimulation alone. Moreover, the addition of increasingdoses of IFN    to A549 cells stimulated with 10 ng of TNF    /mlresulted in a maximal threefold increase in eotaxin mRNA ex-pression over that with TNF    alone (Fig. 4 B  ). Significant syn-ergistic effects of IFN    were also demonstrated in BEAS 2Bcells; these effects were maximal at 6 h and were less apparent18 h after stimulation (data not shown).  Effects of dexamethasone  Pretreatment of A549 cells with varying concentrations of dexa-methasone was associated with a dose-dependent decrease inIL-1    –induced and TNF   -induced eotaxin mRNA expression(Fig. 5,  A  and B ). The presence of dexamethasone resulted infourfold and threefold decreases in IL-1  –induced and TNF  -induced eotaxin mRNA expression, respectively. Dexametha-sone treatment had no effect on cell morphology or viability. Effect of protein-synthesis inhibition on dexamethasone effects When cycloheximide was used to inhibit protein synthesis inA549 cells, eotaxin mRNA was detectable at low levels in theabsence of cytokine stimulation (Fig. 6  A ). Eotaxin mRNA ac-cumulation was superinduced when A549 cells were treatedwith cycloheximide 30 min before IL-1   stimulation; part of the suppressive effect of dexamethasone persisted in the pres-ence of cycloheximide (Fig. 6  A ). TNF  -induced eotaxinmRNA expression was likewise enhanced in the presence of cycloheximide and was less inhibited by dexamethasone thanIL-1  –induced eotaxin mRNA expression (Fig. 6 B ). Eotaxin protein levelsTime course. Eotaxin protein was detected in the absence of stimulation in both the cell supernatant and the cell lysate. Cell Figure 4. Dose-response characteristics of IFN   synergy. Northern analysis of 20  g of total RNA harvested from A549 cells 4 h after stimulation with 0.1 ng/ml IL-1   (  A ) or 10 ng/ml TNF   ( B ) in the presence of increasing concentrations of IFN  . Below each blot a quantitative comparison is made at each dose between the eotaxin-specific signal and the GAPDH-specific signal. Figure 5. Dose-response characteristics for dexamethasone suppres-sion of cytokine-induced eotaxin mRNA accumulation. Northern analysis of 20  g of total RNA harvested from A549 cells 4 h after stimulation with 0.1 ng/ml IL-1   (  A ), or 10 ng/ml TNF   ( B ) and in-creasing concentrations of dexamethasone. Below each blot a quanti-tative comparison is made at each dose between the eotaxin-specific signal and the GAPDH-specific signal. Figure 6. Effects of protein synthesis inhibition on cytokine induction and dexamethasone suppression of eotaxin mRNA accumulation. Northern analysis of 20  g of total RNA harvested from A549 cells4 h after the indicated treatments with 0.1 ng/ml IL-1   (  A ) or 10 ng/ml TNF   ( B ). For cells treated with cycloheximide (10  g/ml) and/or dexamethasone (1  M) plus cytokines, the cycloheximide and dexa-methasone were added 30 min before the addition of the cytokine. Below each blot a quantitative comparison is made with each group between the eotaxin-specific signal and the GAPDH-specific signal. Downloaded from on May 15, 2015.  Eotaxin in Human Airway Epithelial Cells 1771 lysate eotaxin protein levels were significantly increased 2 hafter stimulation with 0.1 ng/ml IL-1  . They were significantlyelevated at all time points measured, and attained their highestlevel 24 h after stimulation (Fig. 7  A ; n     5, P      0.05, com-pared with unstimulated cells). Cell supernatant eotaxin levelswere elevated 24 h after stimulation and were increased fur-ther at 48 h (Fig. 7 B ; n     5, P      0.05, compared with unstimu-lated cells). Dexamethasone effects When 1  M dexamethasone was present in the medium, celllysate levels of eotaxin in groups of cells stimulated 24 h earlierwith 0.1 ng/ml IL-1   were significantly lower than levels incells not exposed to dexamethasone and were not differentfrom levels in cells that had not been stimulated with IL-1  ( n     5, P      0.05). Similarly, the presence of 1  M dexametha-sone resulted in a significant decrease in the level of eotaxin inthe supernatant of cytokine-treated cells at both 24 and 48 h( n     5, P      0.05) compared to cytokine treated cells not ex-posed to dexamethasone. Discussion This study demonstrates that TNF   and IL-1   rapidly andtransiently induce the accumulation of eotaxin mRNA inbronchial epithelial cell lines and that this increase is associ-ated with the intracellular accumulation of eotaxin protein andits secretion into the cell supernatant. These findings, taken to-gether with the known kinetics of TNF   and IL-1   mobiliza-tion in asthmatic airways, the kinetics of eosinophil recruit-ment into human airways after segmental allergen challenge,and the potent eosinophil chemotactic effects of eotaxin, de-fine a mechanism linking cytokine mobilization to eosinophilrecruitment that may be relevant to the pathogenesis of asthma.A growing body of evidence links IL-1   to asthma. LikeTNF  , IL-1   is found at increased levels in the lung lavagefluid of patients with asthma, and in situ hybridization studieshave implicated airway epithelial cells as well as alveolar mac-rophages in IL-1   production (30). These cells are known todisplay surface markers of activation and to produce signifi-cant quantities of IL-1   during episodes of nocturnal asthma ata time when eosinophil recruitment correlates with airflow ob-struction (31). Histological evaluation of lung biopsy speci-mens from patients with nocturnal asthma has documented thepresence of eosinophils predominantly in distal airway seg-ments, where IL-1   produced by alveolar macrophages areavailable to epithelial cells (30). Because ciliated epithelialcells are not present in the distal airway epithelium, the alveo-lar epithelium (as reflected by our findings in alveolar celllines) may be relevant to eosinophil recruitment. Our findingthat IL-1   can act on airway epithelial cells to augment eotaxinmRNA expression, protein production, and secretion supportsthe hypothesis that IL-1   produced by alveolar macrophagesinduces airway epithelial cells to produce eotaxin and may ex-plain the association among IL-1  , airway eosinophilia, andnocturnal airway narrowing.The higher levels of eotaxin immunoreactivity in nasal epi-thelial cells at sites of active allergic inflammation where cy-tokines including TNF   and IL-1   are known to be availableas opposed to adjacent noninflamed, noncytokine-expressingepithelium imply the existence of a mechanism modulating eo-taxin production in allergic airway disease that may involvethese cytokines (20). We found that eotaxin protein secretedfrom A549 cells accumulates over 24 to 48 h in cell superna-tant. Because eosinophil recovery from the asthmatic lung ismaximal 24–48 h after segmental allergen challenge (32, 33)our findings are compatible with the hypothesis that airwayepithelial cell-derived eotaxin contributes to allergen-inducedairway eosinophilia.Although TNF   can cause the characteristic physiologicalphenotype of asthma, namely airway hyperresponsiveness andeosinophilia, the fact that it is not a potent chemoattractant foreosinophils suggests that its effects on eosinophil recruitmentare indirect. Our observation that TNF   significantly enhanceseotaxin mRNA expression at 4 h (22) and the observation thatTNF  -stimulated A549 cells rapidly mobilize a substancechemotactic for eosinophils (34) are compatible with the no-tion that TNF   mobilized by allergic stimulation may contrib-ute to eotaxin production and eosinophil recruitment. A549cells were more sensitive than BEAS 2B cells to TNF  -inducedaugmentation of eotaxin mRNA, but the responsiveness of both cell types suggests that the eotaxin response to cytokinesis not a unique feature of A549 cells. The rapid TNF  -inducedmobilization of eotaxin mRNA is congruent with the timecourse of augmented lung eotaxin mRNA expression follow-ing allergen challenge in animal models (15, 22), while the ac- Figure 7. Eotaxin protein levels. (  A ) Eotaxin protein was measured in A549 cell lysate harvested at the indicated time points after stimu-lation with 0.1 ng/ml IL-1   in the presence and absence of 1  M dexa-methasone ( n     5, * P      0.05). ( B ) Eotaxin protein was measured at the indicated time points following stimulation with 0.1 ng/ml IL-1   in A549 cell supernatant in the presence and absence of 1  M dexa-methasone ( n     5, * P      0.05). Downloaded from on May 15, 2015.
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