Inducible Expression of a Th2-Type CC Chemokine Thymus- and Activation-Regulated Chemokine by Human Bronchial Epithelial Cells1 Takashi Sekiya,*† Misato Miyamasu,* Masako Imanishi,‡ Hirokazu Yamada,* Toshiharu Nakajima,‡ Masao Yamaguchi,* Takao Fujisawa,§ Ruby Pawankar,¶ Yasuyuki Sano,储 Ken Ohta,# Akira Ishii,** Yutaka Morita,** Kazuhiko Yamamoto,* Kouji Matsushima,† Osamu Yoshie,†† and Koichi Hirai2‡ CCR4 is now known to be selectively expressed in Th2 cells. Since the bronchial epithelium is recognized as an important source of mediators fundamental to the manifestation of respiratory allergic inflammation, we studied the expression of two functional ligands for CCR4, i.e., macrophage-derived chemokine (MDC) and thymus- and activation-regulated chemokine (TARC), in bronchial epithelial cells. The bronchial epithelium of asthmatics and normal subjects expressed TARC protein, and the asthmatics showed more intense expression than the normal subjects. On the other hand, MDC expression was only weakly detected in the asthmatics, but the intensity was not significantly different from that of normal subjects. Combination of TNF-␣ and IL-4 induced expression of TARC protein and mRNA in bronchial epithelial A549 cells, which was slightly up-regulated by IFN-␥. The enhancement by IFN-␥ was more pronounced in bronchial epithelial BEAS-2B cells, and a maximum production occurred with combination of TNF-␣, IL-4, and IFN-␥. On the other hand, MDC was essentially not expressed in any of the cultures. Furthermore, expressions of TARC protein and mRNA were almost completely inhibited by glucocorticoids. These results indicate that the airway epithelium represents an important source of TARC, which potentially plays a role via a paracrine mechanism in the development of allergic respiratory diseases. Furthermore, the beneficial effect of inhaled glucocorticoids on asthma may be at least in part due to their direct inhibitory effect on TARC generation by the bronchial epithelium. The Journal of Immunology, 2000, 165: 2205–2213.
D
uring development of an immune response, naive resting CD4⫹ T cells are polarized to either Th1 or Th2 phenotype. According to the Th1/Th2 paradigm (1), inflammation associated with allergic disorders is regarded as a Th2dominant immune response. The cytokines liberated by allergenreactive Th2 cells control the process leading to allergic inflammation; IL-4 plays a primary role in class switching of B cells toward IgE production (2), while IL-5 in proliferation/differentiation of eosinophil lineage (3). In fact, prominent accumulation of lymphocytes of the Th2 phenotype is observed at sites of allergic inflammation (4, 5). Originally, Th1 and Th2 subsets were
Departments of *Allergy and Rheumatology, ‡Bioregulatory Function, †Molecular Preventive Medicine and Core Research for Evolutional Science and Technology (CREST), and **Respiratory Medicine, University of Tokyo Graduate School of Medicine, Tokyo, Japan; §Department of Pediatrics, National Mie Hospital, Mie, Japan; ¶Department of Otolaryngology, Nippon Medical School, Tokyo, Japan; 储Division of Allergy and Respiratory Medicine, Doai Memorial Hospital, Tokyo, Japan; # Department of Internal Medicine, Teikyo University School of Medicine, Tokyo, Japan; and ††Department of Bacteriology, Kinki University School of Medicine, Osaka, Japan Received for publication February 14, 2000. Accepted for publication May 30, 2000. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by a grant from the Manabe Medical Foundation; grantsin-aid from the Ministry of Education, Science, Sports, and Culture of Japan (to M.M., A.I., M.Y., and K.H.); and grants-in-aid from the Ministry of Health and Welfare of Japan (to K.H.). M.M. is a Research Fellow of the Japan Society for the Promotion of Science. 2 Address correspondence and reprint requests to Dr. Koichi Hirai, Department of Bioregulatory Function, University of Tokyo Graduate School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. E-mail address:
[email protected]
Copyright © 2000 by The American Association of Immunologists
functionally separated based on their profiles of cytokine production. However, it has now become apparent that certain types of chemokine receptors are selectively expressed in either Th1 or Th2 cells. Th1 cells express CCR5 and CXC chemokine receptor 3, whereas Th2 cells express CCR3, CCR4, and CCR8 (6 – 8). In vitro studies have revealed that high affinity ligands for CCR4, i.e., macrophage-derived chemokine (MDC)3 (9) and thymus- and activation-regulated chemokine (TARC) (10), induce selective migration of Th2 cells (8). Although there are arguments that CCR4 expression is more related to cutaneous lymphocyte Ag-positive skin-seeking memory T cells than Th2 cells (11), and Th0 cells capable of producing both Th1 and Th2 cytokines may also express CCR4, accumulated evidence to date strongly supports that both MDC and TARC play dominant roles in Th2-type disease conditions such as atopic dermatitis and bronchial asthma (12–14). Bronchial epithelium lines the mucosal surface of the airways, forming a mechanical barrier that separates the external environment from the internal milieu. It has been long believed that the function of epithelial cells is limited to protecting against invading microorganisms and removing particulate matters by means of the mucociliary stairway. Recently, however, substantial evidence has emerged that indicates that airway epithelial cells are able to liberate a number of chemokines fundamental to both inflammatory and immune responses (15–18). Thus, through a paracrine mechanism, chemokines secreted by bronchial epithelial cells may be
3 Abbreviations used in this paper: MDC, macrophage-derived chemokine; DEX, dexamethasone; GCCs, glucocorticoids; MCP, monocyte chemoattractant protein; TARC, thymus- and activation-regulated chemokine.
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TARC EXPRESSION BY BRONCHIAL EPITHELIAL CELLS
involved in the initiation and progression of inflammation of various types. Both IL-8 and monocyte chemoattractant protein (MCP)-1 liberated by airway epithelial cells have been implicated in the initiation and prolongation of acute airway inflammation (15, 16). Epithelial cells are also considered to participate in amplifying respiratory allergic disorders (17, 18). Indeed, epithelial cells of patients with allergic respiratory diseases are likely to be more activated than those of normal individuals in terms of their liberation of inflammatory mediators. Recently, the pathogenic role of airway epithelial cells in airway allergic inflammation has become clear since these cells have been identified as a potent source of eosinophil-specific chemokines such as eotaxin (19), RANTES (20), and MCP-4 (21). Given the potential importance of bronchial epithelial cells in the pathogenesis of respiratory allergic diseases, we investigated production of MDC and TARC, the ligands for the Th2-type chemokine receptor CCR4 (6 – 8), by bronchial epithelial cells. We report in this study that bronchial epithelial cells represent an important cellular source of TARC, which is potentially responsible for the recruitment of Th2 cells in allergic airway disorders.
then frozen and sectioned with a cryostat. The sections were fixed in acetone at ⫺20°C for 10 min and stored at ⫺80°C. After incubation with a blocking solution (TBS containing 2% BSA and 5% normal goat serum), sections were incubated with rabbit anti-human TARC polyclonal Ab (IgG; PeproTech) or primary rabbit anti-human MDC polyclonal Ab (IgG; PeproTech) or control rabbit IgG (Dako, Carpenteria, CA). They were then incubated with biotinylated goat anti-rabbit IgG (Dako) and washed twice, and afterward, sections were incubated with alkaline phosphatase-conjugated streptavidin (Nichirei, Tokyo, Japan). The enzyme activity was visualized with New Fuchsin solution (Dako) containing 5 mM levamizole (Sigma, St. Louis, MO) as a substrate, and finally slides were counterstained with Methyl Green. Immunoreactivity for TARC and MDC was evaluated by two examiners independently of each other and without knowledge of the disease group. The following classification was used to score the staining intensity: 0, negative; 1, weakly positive; 2, moderately positive; 3, strongly positive.
Cell culture A549 cells, a human type II bronchial epithelial cell line, were purchased from Dainippon Pharmaceutical and cultured in Ham’s F12K medium (Sigma) supplemented with 10% FCS and antibiotics. These cells were cultured in 24- or 96-well culture plates (Iwaki Glass, Chiba, Japan), as described previously (24). BEAS-2B cells, a virus-transformed human bronchial epithelial cell line, were obtained from American Type Culture Collection (Manassas, VA) and cultured in LHC-8 medium (Biofluids, Rockville, MD). The BEAS-2B cells were plated on 24-well culture plates (Iwaki Glass) in DMEM/Ham’s F12K medium containing 5% of FCS, as described previously (25). Before stimulation, all of the culture medium in each well was replaced by an identical formulation containing 0.1% BSA in place of FCS.
Materials and Methods Reagents The reagents used in the experiments were the same as described previously (22). Recombinant human TNF-␣, IL-4, and IFN-␥ were purchased from Dainippon Pharmaceutical (Osaka, Japan), PeproTech (Rocky Hill, NJ), and Shionogi Pharmaceutical (Osaka, Japan), respectively.
Assay of chemokines Immunoreactive TARC was quantitated by a double-ligand immunoassay. Samples and standards were incubated overnight in flat-bottom 96-well microtiter plates (Maxisorp; Nunc, Roscilide, Denmark) precoated with 0.5 g/well of an anti-TARC mAb (R&D Systems, Minneapolis, MN) in 0.1 M carbonate buffer, pH 9. After washing, rabbit anti-TARC polyclonal Ab (PeproTech) was added to the plates and reacted for 3 h at room temperature. The plates were then washed, HRP-conjugated mouse anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA) was added, and the enzyme activity was finally determined using tetramethyl-benzidine as a substrate. This ELISA method detected TARC concentrations of greater than 30 pg/ml and did not detect MDC (30 ng/ml). Immunoreactive MDC was quantitated in a similar manner, except that an anti-MDC mAb (R&D Systems) and a rabbit anti-MDC polyclonal Ab (PeproTech) were used as the first and second Abs, respectively. This ELISA method detected MDC concentrations of greater than 30 pg/ml and did not detect TARC (30
Immunohistochemistry Asthmatic subjects and nonatopic normal volunteers were recruited from the outpatients of Doai Memorial Hospital (Table I). Three normal controls demonstrated normal airway reactivity (PC20 histamine ⱖ 20 mg/ml) and no history of wheezing. The studied asthmatic subjects were diagnosed and clinically classified as mild or moderate based on the reported criteria (23). None of them had received systemic corticosteroids for at least 1 mo before the study. Atopy was demonstrated by a positive skin test or detection of allergen-specific IgE for one or more aeroallergens such as house dust mite. The bronchoscopic study was approved by the ethics committee of Doai Memorial Hospital, and all subjects provided written informed consent. Three or more biopsy specimens were obtained from the segmental division of the right upper lobe and the lower lobar carinae using separate forceps. The biopsy specimens were immersed in embedding medium, and
Table I. Demographic, clinical, and immunohistological characteristics of individuals examined in this study
Subject
Age (years)
Sex
Atopic Status
Normal control
1 2 3
30 38 29
M M M
– – –
Asthmatic subjects
1 2 3 4 5 6 7 8 9
42 25 64 55 31 34 48 34 39
M F M M F F M F F
Atopic Atopic Atopic Nonatopic Atopic Atopic Atopic Atopic Atopic
a
Severity
– – –
Inhaled Steroid (g/day)
– – –
Mild None Moderate BDP (1200)d Moderate None Mild None Moderate BDP (1200) Moderate BDP (800) Mild None Moderate FP (400)e Moderate BDP (900)
Positive Skin Reactiona
FEV1.0 (L) (% predicted)
Total IgE (IU/ml) c
Blood Eosinophils (/l)
None None None
– – –
NT 38 12
38 70 77
HD, W HD HD None HD HD, W HD HD HD
3.00 (94.6) 3.25 (98.5) 1.49 (65.4) 2.72 (112.9) 1.40 (53.0) 2.05 (76.5) 2.62 (82.1) 3.45 (140.8) 2.62 (106.5)
152 610 667 161 70 1062 60 75 3306
342 41 1375 365 242 353 122 135 165
Bronchial Epithelial Staining Intensityb TARC
MDC
2 2 2
1 2 1
3 2 3 3 3 3 NT NT NT
1 1 1 NT NT NT 2 1 2
Skin prick test was done using airborne allergen solutions including house dust mite (HD) and weed pollens (W). Intensity of bronchial epithelial immunoreactivity was graded to 0 (no staining), 1 (weak), 2 (moderate), or 3 (strong), as described in Materials and Methods. NT, Not tested. d BDP, Beclomethasone dipropionate. e FP, Fluticasone propionate. b c
The Journal of Immunology ng/ml). Neither of these methods cross-reacted with 30 ng/ml each of MCP-1, MCP-3, RANTES, macrophage-inflammatory protein-1␣, pulmonary and activation-regulated chemokine, liver and activation-regulated chemokine, eotaxin, eotaxin-2, single C motif-1␣, single C motif-1, secondary lymphoid-tissue chemokine, IFN-␥-inducible protein-10, fractalkine, growth-regulated oncogene, I309, IL-8, or stromal cell-derived factor-1␣.
Nonradioisotopic Northern blot analysis Cultured A549 or BEAS-2B cells were grown to confluence in petri dishes and stimulated with cytokines for various time periods. The total cellular RNA was isolated from A549 or BEAS-2B by SNAP Total RNA Isolation Kit. After denaturation, 20 g of total RNA was electrophoresed on 0.41 M formaldehyde/1.2% agarose gels and transferred onto Hybond-N⫹ membranes (Amersham Pharmacia Biotech, Buckinghamshire, U.K.) using TURBOBLOTTER (Schleicher & Schuell, Keene, NH). Hybridization was performed by AlkPhos Direct (Amersham) according to the manufacturer’s instructions. As a TARC DNA probe, a NotI/SalI fragment containing human TARC open reading frame was digested from pSPORT-1/TARC and directly labeled with thermostable alkaline phosphatase. After overnight hybridization at 58°C, the membrane was washed three times with primary wash buffer and twice with secondary wash buffer. The signal was visualized with CDP-Star detection reagent (Amersham) on Hyperfilm ECL (Amersham) and quantitatively analyzed by the BIO-PROFIL densitometry (Vilber Lourmat, Marne La Vallee, France). The membrane was reprobed with a G3PDH probe and the results were standardized.
Statistical analysis Data are presented as the mean ⫾ SEM. Statistical significance of differences was assessed by Student’s t test (paired).
Results Immunohistochemistry for TARC and MDC in bronchial epithelium Frozen tissue sections of biopsy specimens of bronchial mucosa were prepared from normal and asthmatic subjects, and stained with polyclonal Abs against TARC and MDC. We examined three normal volunteers for TARC and MDC, five atopic and one nonatopic asthmatics for TARC, and six atopic asthmatics for MDC. The clinical background and results of immunohistochemical analyses are summarized in Table I, and representative photos are shown in Fig. 1. We observed only weak immunoreactivity with anti-TARC Abs in the bronchial epithelium of the normal subjects, whereas strong staining was clearly seen in the asthmatic bronchial
FIGURE 1. Immunohistochemistry for TARC and MDC in bronchial epithelium. Lung sections obtained from normal subjects and asthmatics were stained with polyclonal Abs against TARC and MDC. A representative of three (normals) and six (asthmatics) different experiments is shown (original magnification: ⫻400).
2207 epithelium (Fig. 1). A statistically significant difference in the intensity was observed between the normals and asthmatics ( p ⫽ 0.025, nonparametric Mann-Whitney U test). In both the normal and asthmatic subjects, TARC immunoreactivity was most prominent in the apical part of bronchial epithelial cells, and the basal cells were also weakly stained. On the other hand, MDC expression was only weakly detected, and no statistically significant difference was observed between the asthmatic and normal subjects (Table I and Fig. 1).
Production of TARC and MDC protein by cytokine-treated bronchial epithelial cell lines In the next series of experiments, we studied the ability of bronchial epithelial cells to generate TARC and MDC in vitro, by using well-accepted human bronchial epithelial cell lines. A549 and BEAS-2B cells were exposed to TNF-␣, IL-4, and IFN-␥, or a combination of these cytokines for 72 h, and both TARC and MDC in the supernatants were quantified by specific ELISA. As shown in Table II, A549 cells generated ng/ml quantities of TARC upon costimulation with TNF-␣ and IL-4, although TNF-␣ or IL-4 alone exerted only marginal effects. IFN-␥ slightly enhanced the TARC production induced by TNF-␣ alone and by TNF-␣ plus IL-4. The pattern of TARC production in BEAS-2B cells was different from that in A549 cells: appreciable amounts of TARC were generated by stimulation with TNF-␣ plus IFN-␥, and by TNF-␣ plus IL-4. Costimulation with these three cytokines synergistically enhanced the TARC level. On the other hand, no detectable amounts of MDC were released by A549 cells or BEAS-2B cells in the same cultures (Table I). The dose-dependent effects of IL-4, IL-13, and IFN-␥ on TARC generation were shown in Figs. 2 and 3. As seen in Fig. 2, A and B, 0.01 and 0.1 ng/ml of IL-4 were sufficient to induce statistically significant TARC production in TNF-␣-stimulated A549 and TNF-␣/IFN-␥-stimulated BEAS-2B cells, respectively. IL-13, which shares a common receptor subunit and diverse biological activities with IL-4, also dose dependently induced TARC generation in these cells (Fig. 2, C and D). When the effects of IFN-␥ on BEAS-2B cells were tested in the presence of fixed doses of IL-4
2208
TARC EXPRESSION BY BRONCHIAL EPITHELIAL CELLS Table II. Comparison of TARC and MDC generation by bronchial epithelial cellsa Cells
A549
BEAS-2B
Stimulus
– TNF IL-4 IFN-␥ TNF/IL-4 TNF/IFN-␥ TNF/IL-4/IFN-␥ LPS – TNF IL-4 IFN-␥ TNF/IL-4 TNF/IFN-␥ TNF/IL-4/IFN-␥ LPS IL-4/IFN-␥
50 ng/ml 10 ng/ml 300 U/ml
1 g/ml 50 ng/ml 10 ng/ml 300 U/ml
1 g/ml
TARC (pg/ml)
MDC (pg/ml)
⬍30
⬍30 5217 ⫾ 367** 221 ⫾ 34** 7118 ⫾ 874* ⬍30
⬍30 ⬍30 ⬍30 ⬍30 ⬍30 ⬍30 ⬍30 ⬍30
36 ⫾ 4 36 ⫾ 4 99 ⫾ 62 36 ⫾ 4 91 ⫾ 37 203 ⫾ 38* 2209 ⫾ 568* 42 ⫾ 6 85 ⫾ 0
⬍30 ⬍30 ⬍30 ⬍30 ⬍30 ⬍30 ⬍30 ⬍30 ⬍30
63 ⫾ 16 59 ⫾ 18
a Cells were stimulated for 72 h as indicated, and both TARC and MDC proteins in the supernatants were assayed by ELISA. Each value represents mean ⫾ SEM (n ⫽ 4). *, p ⬍ 0.05; **, p ⬍ 0.01; significantly different from control (without stimuli), using Student’s t test.
and TNF-␣, statistically significant up-regulation was observed at 1 U/ml of IFN-␥, and the augmentation was maximal at 100 U/ml of IFN-␥ (Fig. 3B). IFN-␣, but not IFN-, enhanced TARC production with statistical significance. The same dose-dependent effect of IFN-␥ was observed in TNF-␣/IL-4-induced TARC production by A549 cells (Fig. 3A). To determine the effect of sequential addition of cytokines, we performed washing experiments (Table III). Both A549 and BEAS-2B cells were stimulated with a first cytokine(s) for 30 min. Then, with and without subsequent washing, the cells were further stimulated with a second cytokine(s) for 48 or 72 h. Elimination of both TNF-␣ and IL-4 by washing resulted in almost complete attenuation of TARC generation, indicating that TARC production requires continuous exposure to TNF-␣ and IL-4. On the other hand, the effect of IFN-␥ did not completely disappear by elimination, indicating that even short-term exposure to IFN-␥ (i.e., 30 min) is sufficient to enhance (A549) or induce (BEAS-2B) TARC generation.
FIGURE 2. Dose-dependent effects of IL-4 and IL-13 on TARC production by bronchial epithelial A549 and BEAS-2B cells. In the presence or absence of graded doses of IL-4 (A and B) or IL-13 (C and D), A549 (A and C) and BEAS-2B (B and D) cells were stimulated with TNF-␣ (50 ng/ml) and TNF-␣ plus IFN-␥ (300 U/ml) for 72 h, respectively. At the end of the incubation period, immunoreactive TARC in each supernatant was determined by ELISA. Bars represent the SEM (n ⫽ 4). ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01, vs control production in the absence of IL-4 or IL-13.
Time kinetics of TARC production We next studied the time kinetics of TARC production at both the mRNA and protein levels. Although no detectable amounts of TARC mRNA were found in unstimulated A549 cells, costimulation with TNF-␣ plus IL-4 strongly promoted the mRNA accumulation in a time-dependent fashion. The level of TARC mRNA was increased within 4 h, drastically up-regulated at 24 h, and maintained at high levels even at 48 h after stimulation. The addition of IFN-␥ clearly up-regulated the accumulation of TARC mRNA induced by TNF-␣ plus IL-4 at 24 and 48 h after stimulation (Fig. 4A). A different pattern of TARC mRNA accumulation was observed in BEAS-2B cells (Fig. 4B). Although TARC mRNA accumulation was hardly detected in BEAS-2B cells stimulated with TNF-␣ plus IL-4 and TNF-␣ plus IFN-␥, stimulation with the combination of TNF-␣, IFN-␥, and IL-4 induced a significant accumulation of TARC mRNA, reaching maximal expression at 24 h of stimulation.
The Journal of Immunology
2209 The time kinetics pattern of TARC protein expression paralleled that of TARC mRNA. As shown in Fig. 5A, stimulation of A549 cells with TNF-␣ plus IL-4 resulted in time-dependent induction of TARC synthesis: the level of TARC increased during 72 h of incubation. Addition of IFN-␥ enhanced the TNF-␣/IL-4-mediated TARC production. On the other hand, stimulation of BEAS-2B cells with the combination of TNF-␣, IL-4, and IFN-␥ resulted in increased TARC production until 48 h of stimulation, but no further liberation of TARC protein was observed after this time point (Fig. 5B). Glucocorticoids (GCCs) inhibit TARC production by bronchial epithelial cell line cells In the last series of experiments, we studied the effects of GCCs on TARC production. As shown in Fig. 6, dexamethasone (DEX) inhibited TARC production by both A549 and BEAS-2B cells in a dose-dependent fashion: half-maximal inhibition was observed over a concentration range from 10⫺9 to 10⫺8 M and in a dosedependent fashion between 10⫺10 and 10⫺7 M. Hydrocortisone (10⫺7 M) also inhibited TNF-␣/IL-4-induced TARC production in A549 cells, while a sex steroid, -estradiol (10⫺7 M), did not inhibit TARC production at all (data not shown). When TARC transcripts in A549 cells were determined by Northern blotting, accumulation of TARC mRNA was dose dependently inhibited by DEX; almost complete attenuation was observed in cells treated with 10⫺8 M DEX (Fig. 7).
Discussion
FIGURE 3. Effects of IFNs on TARC production by bronchial epithelial A549 and BEAS-2B cells. In the presence or absence of graded doses of IFNs, A549 (A) and BEAS-2B (B) cells were stimulated with TNF-␣ (50 ng/ml) and TNF-␣ plus IL-4 (10 ng/ml), respectively. After 72 h, immunoreactive TARC in the supernatants was assayed by ELISA. Bars represent the SEM (n ⫽ 4). ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01, vs control production in the absence of IFNs.
Recent identification of CCR4 as a chemokine receptor selectively expressed in Th2 cells (6 – 8) strongly suggests that TARC and MDC, the ligands for CCR4, are key chemokines in the migration of Th2 cells to the sites of inflammation associated with allergic disorders (8 –10). In fact, results of mouse experiments clearly indicate that neutralization of MDC results in attenuation of pulmonary allergic inflammation (13), while neutralization of TARC leads to inhibition of hepatic Th2-dominant responses (26). Furthermore, a recent report using NC/Nga mice spontaneously developing atopic dermatitis-like lesions indicates possible involvement of TARC in the pathogenesis of atopic dermatitis (14). In contrast to murine T cells, human Th cells may not be exclusively polarized to either Th1 or Th2 phenotypes (27). Nevertheless, TARC and MDC potentially play roles in Th2-dominant conditions in humans as well as in mice.
Table III. Effects of washing on TARC generation by cytokines TARC (pg/ml)a Washing (⫹)
A549
Washing (–)
First
Second
48 h
72 h
48 h
72 h
– TNF-␣ IL-4 TNF-␣ ⫹ IL-4 TNF-␣ ⫹ IL-4 ⫹ IFN-␥ IFN-␥ TNF-␣ ⫹ IL-4
– IL-4 TNF-␣ – – TNF-␣ ⫹ IL-4 IFN-␥
⬍50 ⬍50 ⬍50 ⬍50 ⬍50 4152 ⫾ 314* 60 ⫾ 43
⬍50 81 ⫾ 11 69 ⫾ 37 ⬍50 ⬍50 4886 ⫾ 301† 50
⬍50 1869 ⫾ 127 1862 ⫾ 48 2258 ⫾ 277* 5269 ⫾ 259 4093 ⫾ 124 4209 ⫾ 272
⬍50 2809 ⫾ 338 2560 ⫾ 369 3301 ⫾ 225† 8058 ⫾ 861 6595 ⫾ 824 7896 ⫾ 924
– TNF-␣ ⫹ Il-4 TNF-␣ ⫹ IL-4 ⫹ IFN-␥ IFN-␥ TNF-␣ ⫹ IL-4
– – – TNF-␣ ⫹ IL-4 IFN-␥
⬍50 ⬍50 ⬍50 567 ⫾ 105‡ ⬍50
h⬍50 ⬍50 ⬍50 564 ⫾ 218§ ⬍50
⬍50 ⬍50‡ 1043 ⫾ 189 826 ⫾ 224 890 ⫾ 117
⬍50 89 ⫾ 12§ 1000 ⫾ 201 988 ⫾ 259 1141 ⫾ 114
BEAS-2B
a Each value represents the mean ⫾ SEM (n ⫽ 4). *, p ⬍ 0.05;†, p ⬍ 0.01; ‡, p ⬍ 0.01; §, p ⬍ 0.05; between the two values.
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TARC EXPRESSION BY BRONCHIAL EPITHELIAL CELLS
FIGURE 4. Time kinetics of TARC mRNA induction by bronchial epithelial by A549 and BEAS-2B cells. A549 (A) and BEAS-2B (B) were treated with TNF-␣ (50 ng/ml) plus IL-4 (10 ng/ml) in the absence or presence of IFN-␥ (300 U/ml) for the indicated times, and the total cellular RNA was extracted for Northern blot analysis. A representative blot (20 g total RNA/lane) was hybridized with TARC or G3PDH cDNA probe. The intensity of the bands was quantitated by densitometric scanning. Data are shown as the steady state levels of TARC mRNA expression divided by the level of G3PDH mRNA expression. A representative of two different experiments is shown, and another showed similar results.
To date, a number of cellular sources of TARC and MDC have been identified, which include CD4-positive T cells, macrophages, and dendritic cells (8, 9). In the present study, we have evaluated the ability of bronchial epithelial cells to express and produce TARC at both the mRNA and protein levels. Immunohistochem-
ical studies revealed intense positive staining of the bronchial epithelium by anti-TARC Abs in asthmatic subjects. On the other hand, MDC expression was only weakly detected, and no significant difference of intensity was observed between the asthmatics and normal subjects. Results of in vitro experiments demonstrated
FIGURE 5. Time kinetics of TARC protein production by bronchial epithelial by A549 and BEAS-2B cells. A, Time kinetics of TARC production by A549 cells. A549 cells were stimulated with TNF-␣ (Œ, 50 ng/ml), IL-4 (‚, 10 ng/ml), IL-4 plus TNF-␣ (E), TNF-␣ plus IL-4 plus IFN-␥ (F, IFN-␥, 300 U/ml), or control medium (䊐). After the indicated time periods, the level of TARC protein was assayed by ELISA. Bars represent the SEM (n ⫽ 4). B, Time kinetics of TARC production by BEAS-2B cells. BEAS-2B cells were stimulated with TNF-␣ (10 ng/ml) plus IL-4 (10 ng/ml, E), TNF-␣ plus IL-4 plus IFN-␥ (F, IFN-␥, 300 U/ml) for various time periods. After the indicated time periods, the level of TARC protein was assayed by ELISA. Bars represent the SEM (n ⫽ 4). ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01, vs TARC production in the absence of IFN-␥.
The Journal of Immunology
FIGURE 6. Inhibition of TARC protein production by DEX. In the presence of graded doses of DEX (F) or control DMSO (E), A549 (A) and BEAS-2B (B) cells were stimulated with TNF-␣ (50 ng/ml)/IL-4 (10 ng/ ml), and TNF-␣/IL-4/IFN-␥ (300 U/ml), respectively. After 72 h, the immunoreactive TARC in the supernatants was assayed by ELISA. Values are expressed as the percentage of control production initiated by each stimulus in the absence of DEX. Bars represent the SEM (n ⫽ 4). ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01, vs control production in the absence of DEX.
FIGURE 7. Inhibition of TARC mRNA expression by DEX. A549 cells were stimulated with TNF-␣ (50 ng/ml) plus IL-4 (10 ng/ml) in combination with graded doses of DEX, after which the total cellular RNA (20 g total RNA/lane) was extracted for Northern blot analysis. A representative of two different experiments is shown, and another showed similar results.
2211 that bronchial epithelial cell lines were capable of generating significant quantities of TARC protein, comparable with the reported production by monocytes on a single cell basis (8). The in vitro secretion of TARC by bronchial epithelial cell lines strongly suggests the in vivo elaboration of a Th2-specific chemokine from the bronchial epithelium, which potentially contributes to the accumulation of Th2 cells in the airway. Because of the anatomical location of bronchial epithelial cells in the airway system, TARC liberated by these cells functions as a paracrine factor to attract Th2 cells to the respiratory tract. In human monocytes/dendritic cells, TARC production was shown to be up-regulated by a Th2-derived cytokine, IL-4 (8). Our present studies also found IL-4 to be essential for TARC generation by epithelial cells. IL-4 by itself exerted only marginal effects, but it strongly potentiated TNF-␣-induced TARC production by A549 cells and TNF-␣/IFN-␥-induced TARC production by BEAS-2B cells. Furthermore, IL-13, which shares a common receptor subunit with IL-4 (28), also enhanced TNF-␣-mediated TARC production by A549 cells. To date, a substantial body of evidence links IL-4 and IL-13 to allergic disorders: both cytokines induce class switching toward IgG4 and IgE (2), and they also selectively induce VCAM-1 expression on venous endothelial cells, which may contribute to the recruitment of eosinophils (29, 30). In addition, IL-4 and IL-13 up-regulate the expression of an eosinophil-specific chemokine, eotaxin, in various types of cell, including bronchial epithelial cells (25, 31). Our present findings that IL-4 and IL-13 have promoting effects on TARC production by bronchial epithelial cells further extend the proinflammatory roles of these molecules in respiratory allergic diseases. Since both IL-4 and IL-13 are selectively produced by T cells of the Th2 phenotype, these findings suggest the existence of a positive loop of regulation for additional Th2 recruitment in allergic diseases. A Th1-derived cytokine, IFN-␥, also stimulated TARC production: TARC protein synthesis as well as TARC mRNA expression was markedly up-regulated by IFN-␥. The exquisite sensitivity to IFN-␥ indicates that the enhancing action was exerted via interaction with specific receptors. These results were rather unexpected, because allergic reactions are usually postulated in the context of a Th2-type cytokine response, not a Th1-type response. Furthermore, it has been demonstrated that production of MDC by monocytes is down-regulated by IFN-␥ (32). Although Th1 cytokines are generally assumed to be against the development of allergic symptoms, IFN-␥ may exert dichotomous effects on allergic reactions. IFN-␥ inhibits the development of Th2 cells, and antagonizes IL-4-dependent effects such as IgE synthesis (33). Furthermore, studies performed in experimental animals demonstrated that IFN-␥ suppressed the development of eosinophilic inflammation (34). On the other hand, under certain circumstances, especially in cases suffering from a viral infection, IFN-␥ possibly plays a promoting role in allergic inflammation. In vivo mouse experiments demonstrated that preceding infection with respiratory syncytial viruses enhanced the response to subsequent sensitization with Ags (35). The viral infection alone was found to induce a Th1 cytokine. But importantly, when sensitized mice were infected with a virus and exposed to the corresponding Ag, they showed a strong Th2 cytokine response, resulting in augmentation of the allergic response (35). In our experiments, IFN-␥ clearly up-regulated TARC production, suggesting that the reaction to a viral infection in a host with bronchial asthma may promote a Th2 cytokine response instead of a Th1 cytokine response. GCCs have long been extensively used to treat allergic disorders, with remarkable success (36). For patients with mild, moderate, and severe asthma, inhaled GCCs now represent the first line of medication. Although not fully proven, inhibition of elaboration
2212 of chemokines fundamental to allergic inflammation may be one of the mechanisms by which GCCs exert potent antiallergic effects (17, 37). In fact, previous reports by others have revealed that in vitro generation of eosinophil-directed chemokines, including eotaxin (19), RANTES (20), and MCP-4 (21), by bronchial epithelial cells is completely attenuated by GCCs. In the present study, we have demonstrated that treatment with various GCCs resulted in attenuation of TARC mRNA expression and concomitant loss of the ability to produce TARC protein. The airway epithelium is the first cell layer to be encountered by inhaled GCCs, indicating that bronchial epithelial cells are potential targets for this drug. Since bronchial epithelium-derived TARC potentially contributes to allergic inflammation via a paracrine mechanism, the beneficial effect of inhaled GCCs in bronchial asthma is due at least in part to the direct inhibitory effect of GCCs on TARC generation by bronchial epithelial cells. The present results clearly extend our previous knowledge of the antiinflammatory roles of GCCs in the regulation of allergic diseases. Even though the importance of CCR4 ligands in pathogenesis of Th2-type disease conditions has been increasingly accepted, it remains mostly obscure how TARC and MDC play roles in bronchial asthma. Although bronchial epithelial cells seem to be a dominant source of TARC, but not MDC, alveolar macrophages, another important source of chemokines in the airways, potentially generate large amounts of MDC. In fact, strong MDC expression was induced in alveolar macrophages in experimentally induced allergic reactions in mice (13). Thus, further studying regarding protein levels of both chemokines in bronchoalveolar lavage fluid or induced sputum from asthmatic patients will be useful to address such important issues. In summary, we, for the first time, have demonstrated that bronchial epithelial cells represent an important source of a Th2-specific chemokine, TARC. TARC production was up-regulated not only by Th2-derived cytokines IL-4 and IL-13, but also by a Th1derived cytokine, IFN-␥. The TARC promoter contains consensus recognition sequences for both IFN-␥ response elements and STAT-6. Because regulation of TARC generation has therapeutic potential for the treatment of allergic airway disorders, we are currently investigating transcriptional regulation of the TARC promoter.
Acknowledgments We thank S. Takeyama for her excellent secretarial help.
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