IL-13 and IL-4 Up-Regulate Cysteinyl 1 Expression in Human Monocytes and Macrophages1

Maryse Thivierge, Jana Stanˇkova«, and Marek Rola-Pleszczynski2

The cysteinyl (Cys) (LT)C4, LTD4, and LTE4, are lipid mediators that have been implicated in the pathogenesis of . The human LTD4 receptor (CysLT1R) was recently cloned and characterized. The present work was undertaken to study the potential modulation of CysLT1R expression by the Th2 cytokines IL-13 and IL-4. In this study, we report that IL-13 up-regulates CysLT1R mRNA levels, with consequently enhanced CysLT1R protein expression and function in human monocytes and monocyte-derived macrophages. CysLT1R mRNA expression was augmented 2- to 5-fold following treatment with IL-13 and was due to enhanced transcriptional activity. The effect was observed after 4 h, was maximal by 8 h, and maintained at 24 h. IL-4, ␥ but not IFN- , induced a similar pattern of CysLT1R up-regulation. Monocytes pretreated with IL-13 or IL-4 for 24 h showed enhanced CysLT1R protein expression, as assessed by flow cytometry using a polyclonal anti-CysLT1R Ab. They also showed 2؉ enhanced responsiveness to LTD4, but not to LTB4, in terms of Ca mobilization, as well as augmented chemotactic activity. Our

findings suggest a possible mechanism by which IL-13 and IL-4 can modulate CysLT1R expression on monocytes and macro- phages, and consequently their responsiveness to LTD4, and thus contribute to the pathogenesis of asthma and allergic diseases. The Journal of Immunology, 2001, 167: 2855Ð2860.

eukotrienes (LTs),3 which are derived through the 5-li- inflammation of the bronchial mucosa. Significantly elevated expres- poxygenase pathway of metabolism, are sion of IL-13 mRNA and protein has been observed in BAL cells of L lipid mediators of inflammation and immediate hypersen- patients with atopic asthma after allergen challenge (10). More re- sitivity (1Ð3). The cysteinyl (Cys)LTs, LTC4, LTD4, and LTE4 cently, blockade of IL-13 before aeroallergen challenge was shown to (components of the slow-reacting substance of anaphylaxis), are be sufficient to attenuate AHR (11). Unlike IL-4, the production of potent lipid mediators implicated mainly in acute bronchoconstric- IL-13 can be sustained through the late asthmatic response, and the tion and chronic airway inflammation in asthma. When instilled concentration of secreted IL-13 strongly correlates with the number of into the airways, they mimic many of the features of human eosinophils in BAL and in bronchial submucosa (12). Together, these asthma, including bronchoconstriction, mucus secretion, and air- results suggest that IL-13 plays an important role in the pathogenesis way hyperresponsiveness (AHR) (1Ð4). Recently, Lynch et al. (5) of asthma. and Sarau et al. (6) reported the cloning and characterization of a IL-13 and IL-4 have several overlapping biological activities ␣ high affinity cell surface human LTD4 receptor (CysLT1R) that (13, 14) that may be due to a shared receptor subunit, IL-4R (15), belongs to the G protein-coupled receptor family. In normal lung, and signaling through a shared STAT6-dependent pathway (16). In

CysLT1R mRNA was found to be expressed in macrophages as addition to potent anti-inflammatory properties on macrophages well as in peribronchial smooth muscle (5). and other cells (17Ð20), both cytokines also induce several immu- The inflammatory pathology associated with asthma is thought to nostimulatory functions, such as increase in VCAM-1 expression be mediated by Th2 lymphocytes and their cytokine network. Nu- on the surface of endothelial cells and induction of monocyte che- merous studies of bronchoalveolar lavage (BAL) and biopsies from moattractant protein-1 (MCP-1) production (21, 22). Because both asthmatic airways have shown an increase in CD4ϩ T lymphocytes LTs and Th2 cytokines are major players in asthma and because producing Th2-like cytokines, IL-4, IL-5, and IL-13 (7Ð9). They have the level of expression of CysLT1R could be expected to affect suggested the importance of these cytokines in AHR, which is one of cellular responses to LTD4, we initiated the present study to in- the defining features of asthma and believed to result from chronic vestigate the potential for IL-13 and IL-4 to modulate the expres-

sion and function of CysLT1R.

Immunology Division, Department of Pediatrics, Faculty of Medicine, Universite«de Sherbrooke, Sherbrooke, Canada Materials and Methods Cells Received for publication March 6, 2001. Accepted for publication June 22, 2001. The costs of publication of this article were defrayed in part by the payment of page Monocytes were obtained from peripheral blood of healthy medication-free charges. This article must therefore be hereby marked advertisement in accordance volunteers, following informed consent in accordance with an Internal Re- with 18 U.S.C. Section 1734 solely to indicate this fact. view Board-approved protocol. Peripheral blood leukocytes were enriched 1 This work was supported by the Canadian Institutes of Health Research, the Fonds by dextran sedimentation, layered over a Ficoll-Hypaque cushion, and cen- de Recherche en Sante« du Que«bec Respiratory Health Network, and the Clinical trifuged at 400 ϫ g for 20 min. Mononuclear leukocytes were collected at Research Center of the Center Hospitalier Universitaire de Sherbrooke. the interface and washed twice with PBS and resuspended in RPMI 1640 2 Address correspondence and reprint requests to Dr. Marek Rola-Pleszczynski, Im- (Life Technologies, Burlington, Ontario, Canada) supplemented with 10% munology Division, Department of Pediatrics, Faculty of Medicine, Universite«de heat-inactivated FBS (Sigma-Aldrich, Oakville, Ontario, Canada), 100 Sherbrooke, 3001, North 12th Avenue, Sherbrooke, J1H 5N4 Quebec, Canada. E-mail U/ml penicillin, and 100 ␮g/ml streptomycin in a humidified atmosphere address: [email protected] with 5% carbon dioxide at 37¡C. Monocytes were then purified by adher- 3 Abbreviations used in this paper: LT, leukotriene; Cys, cysteinyl; AHR, airway ence (60 min, 37¡C) on plastic petri dishes coated with defibrinized autol- ogous serum and removed with EDTA (0.01 M) in RPMI 1640Ð10% FBS. hyperresponsiveness; CysLT1R, LTD4 receptor; BAL, bronchoalveolar lavage; 2ϩ 2ϩ ϫ 6 [Ca ]i, intracellular Ca concentration; JAK, Janus kinase; MCP, monocyte che- Cells were resuspended in RPMI 1640Ð10% FBS at 1 10 cells/ml and moattractant protein; PAF, -activating factor. allowed to rest overnight before stimulation with the appropriate stimuli.

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 2856 IL-13 AND IL-4 INDUCE LEUKOTRIENE D4 RECEPTOR EXPRESSION

Adherent cells cultured on plastic petri dishes for up to 5 days were re- and exposed to Hyperfilm MP with intensifying screens at Ϫ80¡C for 2 wk. ferred to as monocyte-derived macrophages. For immobilization of DNA to membranes, 5 ␮g cDNA was denatured with 0.3 M NaOH at room temperature for 30 min, neutralized with am- Cytokines and reagents monium acetate (final concentration of 1.5 M), and spotted onto nylon Human rIL-4 and rIL-13 were obtained from PeproTech (Rocky Hill, NC); membrane using a slot blot apparatus. ␥ human IFN- was obtained from R&D Systems (Minneapolis, MN); all Flow cytometry cytokine preparations contained Ͻ0.1 ng endotoxin per microgram (1 EU/ ␮ g); rabbit polyclonal anti-human CysLT1R Ab was developed and char- For flow cytometry studies, cells were washed with PBS and fixed with 2% acterized with Cayman Chemical (Ann Arbor, MI); rabbit IgG isotype paraformaldehyde for 15 min at room temperature, followed by permeabi- control was obtained from Southern Biotechnology Associates (Birming- lization with 0.1% saponin for an additional 15 min at room temperature. ham, AL); FITC-conjugated goat anti-rabbit IgG was obtained from Bio/ Cells were resuspended with PBS-2% BSA and labeled for 30 min at 4¡C

Can Scientific (Mississauga, Ontario, Canada); LTB4, LTD4, and platelet- with anti-CysLT1R Ab or with isotype control Ab. Cells were then washed activating factor (PAF) were obtained from Cayman Chemical; MK571 with cold PBS and incubated for 30 min at 4¡C with FITC-conjugated goat was obtained from Biomol (Plymouth Meeting, PA). anti-rabbit IgG. Finally, cells were washed again and resuspended in PBS before single-color immunofluorescence analysis of 5000 cells was per- Northern blot analysis formed on a FACScan flow cytometer (BD Biosciences, San Jose, CA). Total cellular RNA was extracted by the guanidium thiocyanate method Intracellular calcium mobilization (23), separated by electrophoresis on 1% agarose, and transferred onto a ϩ Hybond-N (Amersham Pharmacia Biotech, Baie d‘Urfe«, Quebec, Can- For Ca2ϩ mobilization assays, 5 ϫ 106 cells were loaded in HBSS (Life ada) membrane for Northern analysis. The cDNA corresponding to the Technologies) containing 350 mg/L NaHCO3 and 10 mM HEPES (pH 7) whole coding sequence of human CysLT1R (5) was amplified by PCR from with the calcium indicator fura 2-AM (Molecular Probes, Eugene, OR) for DNA of human monocytes, using the primers 5Ј-CGGGATCCGATGA 30 min at room temperature. Loaded cells were washed twice, suspended AACAGGAAATC-3Ј as sense and 5Ј-CCGGAATTCAATGGGTTTAAA in fresh loading buffer, and added to a constantly stirred cuvette, main- Ј CTATAC-3 as antisense. The amplified CysLT1R fragment contained tained at 37¡C in a SLM/Aminco spectrofluorometer (SLM Instruments, 1014 bp. Control hybridizations were performed with the human GAPDH Urbana, IL). The concentration of extracellular Ca2ϩ was brought to 1.5 cDNA probe obtained from the American Type Culture Collection (Man- mM by addition of a solution of CaCl2 into the cuvette 10 min before assas, VA). The probes were labeled with a multiprime DNA labeling recordings. Maximal cell fluorescence (Fmax) was obtained by adding Tri- system (Amersham Pharmacia Biotech) using [␣-32P]dCTP (sp. act., 3000 ton X-100 to a final concentration of 0.5%. Minimal fluorescence (Fmin) Ci/mmol; Amersham Pharmacia Biotech). Membranes were prehybridized was determined by subsequent addition of the chelator EGTA in Tris-HCl for4hinamixture containing 120 mM Tris (pH 7.4), 600 mM NaCl, 8 buffer (100 mM (pH 9)) at 125 mM. Stimuli consisted of LTD4, LTB4, mM EDTA (pH 8), 0.1% sodium pyrophosphate, 0.2% SDS, and 100 and PAF. ␮g/ml heparin; hybridization was performed overnight at 60¡C in the same mixture in which the concentration of heparin was increased to 625 ␮g/ml Chemotaxis assay and dextran sulfate at 10% was added. The membranes were then washed ϫ ϫ Monocytic chemotactic activity was performed with Boyden chambers us- once at room temperature for 20 min in 2 SSC (1 SSC: 0.15 M NaCl, ␮ 0.15 M sodium citrate (pH 7)) and once with 0.1ϫ SSC. The membranes ing a modified Boyden chamber chemotaxis assay. A volume of 200 l ϫ 5 were exposed to Hyperfilm MP (Amersham Pharmacia Biotech) with in- cells (6 10 ) in RPMI 1640, supplemented with 2.5 mg/ml BSA, was tensifying screens at Ϫ80¡C. added to the upper chamber. The lower chamber contained graded con- centrations of LTD4 or its vehicle. For chemokinesis studies, both cham- Nuclear run-on transcription assay bers contained LTD4 or its vehicle. The two chambers were separated by a5-␮m-pore-size polycarbonate filter (Osmonics, Westborough, MA). Af- Before nuclei isolation, monocytes were stimulated for 3 or 6 h with IL-13 ter incubation for2hat37¡Cin5%CO2, the chambers were disassembled (10 ng/ml). Following stimulation, cells were washed with ice-cold PBS, and the upper side of the filter was scraped free of cells. Cells on the lower ϫ ϫ 7 and pelleted at 250 g for 5 min. The cell pellet (5 10 cells) was then side were removed with 5 mM EDTA, centrifuged, and resuspended in resuspended in 1 ml lysis buffer (10 mM Tris-HCl (pH 7.4), 10 mM KCl, PBS. An aliquot of 100 ␮l was then counted in the FACScan. 5 mM MgCl2, 0.5% Nonidet P-40 (v/v)), incubated for 5 min on ice, and centrifuged at 500 ϫ g for 5 min at 4¡C. The supernatant was discarded, Results and the nuclei were resuspended in 200 ␮l freezing buffer (50 mM Tris- The expression of CysLT1R in human monocytes was first investi- HCl, 40% glycerol (v/v), 5 mM MgCl2 (pH 8), 0.1 mM DTT) and frozen in liquid nitrogen. For the transcription assay, nuclei were thawed on ice gated by Northern blot analysis of total RNA following8hofstim- and pelleted at 500 ϫ g for30sat4¡C, and the supernatant was discarded. ulation with graded concentrations of IL-13 ranging from 1 to 20 Nuclei were then mixed with 100 ␮l reaction buffer containing 100 mM ng/ml. As shown in Fig. 1A, human monocytes constitutively ex- Tris-HCl (pH 7.9), 300 mM (NH4)2SO4, 4 mM MgCl2, 200 mM NaCl, 0.4 mM EDTA, 1 mM DTT, 40% glycerol, 1 mM each of ATP, CTP, and pressed low levels of CysLT1R mRNA, and IL-13 induced a time- GTP, 1 ␮l RNase inhibitor (40 U/␮l RNasin; Promega, Madison, WI), dependent augmentation of transcript levels. Accumulation was aug- and 150 ␮Ci [␣-32P]UTP (3000 Ci/mmol; Amersham Pharmacia Biotech) mented 3- to 5-fold over baseline, and the effect was detectable as and incubated at 30¡C for 30 min. The 32P-labeled RNA transcripts were soon as4hofstimulation, was maximal at 8 h, and was still detectable then incubated for a further 10 min at 30¡C in the presence of 100 U DNase ␮ at 24 h. IL-13 induced a concentration-dependent augmentation in 1 (10 U/ l, RNase free; Promega) and 0.5 mM CaCl2. This procedure was followed by addition of 1 vol of 2ϫ proteinase K buffer (20 mM Tris-HCl steady state levels of CysLT1R mRNA, which plateaued at 10 ng/ml (pH 7.9), 20 mM EDTA, 1% SDS) and 5 ␮l proteinase K (10 mg/ml) and (Fig. 1B). In parallel experiments, we also examined the effect of the 32 incubation at 42¡C for 30 min. The P-labeled RNA transcripts were then cytokine IL-4 on CysLT1R mRNA expression. Fig. 1C illustrates isolated with saturated phenol solution and chloroform-isoamyl alcohol CysLT R mRNA levels in monocytes and monocyte-derived macro- (49/1), mixed well, incubated on ice for 15 min, and then centrifuged for 1 15 min at 500 ϫ g at 4¡C. The elongated RNA was then purified on a phages following6hofincubation in the absence or presence of Sephadex G-25 column. Before ethanol precipitation, 32P-labeled RNA IL-13 (10 ng/ml) or IL-4 (10 ng/ml). Both cytokines induced similar transcripts were denatured adding NaOH (final concentration of 0.2 M) for levels of CysLT1R mRNA expression in both cell populations. Fig. 15 min on ice. The solution was neutralized by the addition of HEPES, pH 1D illustrates the kinetics of up-regulation of CysLT R mRNA ex- 32 1 5.5 (final concentration of 0.28 M). Elongated P-labeled RNA transcripts pression by IL-4, with a maximal effect at4hofincubation. Fig. 1E were then precipitated adding 1 vol of ethanol and 0.1 vol of 3 M sodium acetate and centrifuged 15 min at 500 ϫ g at 4¡C. The pellet was resus- shows the effect to be concentration dependent, with up-regulation pended in 500 ␮l hybridization solution (0.75 M NaCl, 50 mM HEPES (pH detectable at 0.1 ng/ml IL-4. Moreover, the Th1-type cytokine IFN-␥ ϫ 7), 2 mM EDTA (pH 8), 50% deionized formamide, 0.5% SDS, 10 was not capable of up-regulating CysLT1R. Denhardt’s, and 0.5 mg/ml salmon sperm DNA). RNA was then denatured In a further series of experiments, we evaluated the mechanisms at 90¡C for 5 min and hybridized at 42¡Cfor48hto5␮g denatured DNA immobilized on positively charged nylon transfer membrane (Mandel, underlying the accumulation of CysLT1R mRNA induced by IL-13. Saint-Laurent, Quebec, Canada) in 3 ml hybridization solution. Membranes The augmentation of CysLT1R mRNA accumulation in monocytes were then washed four times at 42¡C for 15 min in 0.1ϫ SSC, 0.1% SDS following IL-13 treatment could result from augmentation in mRNA The Journal of Immunology 2857

FIGURE 1. Up-regulation of CysLT1 receptor gene expression by IL-13 and IL-4 in human monocytes and monocyte-derived macrophages. Northern blot analysis of CysLT1R mRNA expression. A, Human monocytes were incubated in the absence (M) or presence of IL-13 (10 ng/ml) for different times, as indicated. B, Mono- cytes were incubated for 6 h with graded concentrations of IL-13. C, Monocytes or monocyte-derived macro- phages were incubated for 6 h with IL-13 or IL-4 (10 ng/ml). D, Monocytes were incubated for 2Ð8hinthe absence or presence of 10 ng/ml IL-4. E, Monocytes were incubated for 4 h with graded concentrations of IL-4 or with 400 U/ml IFN-␥. Total RNA was purified from monocytes or monocyte-derived macrophages and used in Northern blot analysis, as described in Materials and Methods. Results are representative of at least three separate experiments.

⌬ stability and/or gene transcription rate. We first examined the former IL-13 or IL-4 showed increased calcium flux in response to LTD4 ( 2ϩ 2ϩ ϭ Ϯ possibility by assessing CysLT1R mRNA decay kinetics with medi- intracellular Ca concentration ([Ca ]i) 129.5 34.9 nM for um- and IL-13-treated monocytes using the RNA synthesis inhibitor, control cells vs 251.8 Ϯ 63.7 nM for IL-13-treated cells ( p Ͻ 0.02), actinomycin D (10 ␮g/ml). Monocytes were incubated4hinthe and 244 Ϯ 66.8 nM for IL-4-treated cells ( p Ͻ 0.05), n ϭ 4), but not ⌬ 2ϩ ϭ Ϯ Ϯ presence or absence of IL-13 before addition of actinomycin D, and to LTB4 ( [Ca ]i 111.8 25.5 nM for control cells vs 132.8 Ͼ Ϯ CysLT1R mRNA levels were assessed by Northern blot analysis after 38.2 nM for IL-13-treated cells ( p 0.1), and 149.4 28.4 nM for Ͼ ϭ periods of 0Ð180 min. As illustrated in Fig. 2A, CysLT1R mRNA t1/2 IL-4-treated cells ( p 0.1), n 5), nor to PAF (data not shown). The ϳ was 2.5 h in unstimulated human monocytes. IL-13 treatment did response to LTD4, but not to LTB4, was totally blocked by the se- not induce a significant change in CysLT1R mRNA t1/2, suggesting lective CysLT1R antagonist MK571. As shown in Fig. 4B, respon- that the increased accumulation of CysLT1R mRNA by IL-13 was not siveness to LTD4 in monocyte-derived macrophages was augmented caused by stabilization of the transcripts. To assess whether IL-13 even more than in monocytes, following a 24-h incubation with the induced CysLT1R mRNA accumulation through a transcriptional cytokines IL-13 or IL-4 (10 ng/ml). mechanism, we performed experiments in which monocytes were The functional relevance of increased CysLT1R expression on IL- pretreated with actinomycin D for 15 min to block new RNA syn- 13-stimulated monocytes was also investigated in terms of modula- thesis and were then treated with either medium or IL-13. As shown tion of their chemotactic response to LTD4. Whereas resting mono- in Fig. 2B, this pretreatment completely abolished the IL-13-induced cytes showed a weak chemotactic response to LTD4, Fig. 5A shows accumulation of CysLT1R mRNA, suggesting a transcriptional mech- that a significantly augmented response to LTD4 was observed when anism in the regulation of CysLT1R expression by IL-13. Transcrip- monocytes had been exposed for 24 h to IL-13. Enhanced migration tional activation of the CysLT1R gene was confirmed by a nuclear to LTD4 was concentration dependent, with activity already present at run-on experiment (Fig. 2C) that showed that nuclear transcription of 1 nM and reaching a plateau between 10 and 100 nM LTD4. Similar the CysLT1R gene was indeed increased (4-fold at 3 h) by IL-13 results were obtained with IL-4-treated cells. Chemokinesis was not treatment. affected, however, by pretreatment of cells with either IL-4 or IL-13 The next series of experiments examined whether transcription of (Fig. 5B). Moreover, monocyte chemotactic responses to the chemo-

CysLT1R mRNA was associated with an augmented cellular expres- kine MCP-1 were not affected by such pretreatment (Fig. 5B). These sion of CysLT1R protein. Changes in monocyte mRNA levels were observations suggest that the augmentation of CysLT1R expression paralleled by an increase in cellular expression of CysLT1R, as as- following stimulation with IL-13 or IL-4 is accompanied by an en- sessed by flow cytometry using specific anti-CysLT1R Ab. As illus- hanced functional activity of the receptor. trated in Fig. 3A, both IL-13 and IL-4 induced a markedly augmented expression of CysLT1R protein in monocytes. The effect was maxi- mal following a 24-h stimulation with the cytokines. The effects of the Discussion two cytokines were both concentration and time dependent, being The CysLTs, particularly LTD4 and LTC4, are lipid mediators that maximal at 10 ng/ml after 24 h (Fig. 3, B and C). Similarly to mono- have been implicated in the pathogenesis of several inflammatory pro- cytes, IL-13 and IL-4 also up-regulated the expression of CysLT1Rin cesses, including asthma. Their multiple and diverse cellular actions monocyte-derived macrophages (Fig. 3A). make them also potential protagonists in a number of other patholog- We finally investigated whether the IL-13- and IL-4-induced up- ical states, such as adult respiratory distress syndrome and pulmonary regulation of CysLT1R expression was associated with augmented hypertension. The present work was initiated to study the mechanisms functional responsiveness to LTD4. Receptor function was evaluated by which the receptor for LTD4 can be regulated. Our understanding by mobilization of intracellular calcium upon stimulation with LTD4. of the mechanisms governing the regulation of CysLT1R expression

As illustrated in Fig. 4A, monocytes cultured for 24 h with 10 ng/ml is a prerequisite for attempting to modulate and control CysLT1R 2858 IL-13 AND IL-4 INDUCE LEUKOTRIENE D4 RECEPTOR EXPRESSION

FIGURE 2. Transcriptional regulation of CysLT1 receptor gene expression following stimulation with IL-13. A, Effect of IL-13 (10 ng/ml) on the CysLT1R mRNA stability. Monocytes were pretreated for 4 h with IL-13 before addition of actinomycin D, and CysLT1R mRNA half-life was measured and expressed as percentage of values at time 0. B, Monocytes were pretreated for 15 min with either medium or actinomycin D (10 ␮g/ml) before stimulation with IL-13 (10 ng/ml) for 3 h. Total RNA was extracted and used in Northern blot analysis, as described in FIGURE 3. Flow cytometric analysis of CysLT1R expression in cells stimu- Materials and Methods. C, Nuclear run-on assay. Monocytes incubated in the lated with IL-13 or IL-4. Human monocytes or monocyte-derived macrophages absence or presence of 10 ng/ml IL-13 for 3 or 6 h. Equal loading and transfer were were incubated with IL-13 or IL-4 (10 ng/ml) for 24 h. Cells were subsequently assessed by comparison with GAPDH gene expression. labeled with anti-CysLT1R or isotype-matched control Abs, followed by incuba- tion with FITC-conjugated goat anti-rabbit IgG. A, Results of a single experiment, representative of at least four, are shown. Dotted lines represent labeling with isotype control Ab. Solid thin and thick lines represent labeling of medium- and expression in various tissues in LTD4-mediated diseases, and partic- ularly asthma. In the present study, we have shown that IL-13 and cytokine-treated cells, respectively, with anti-CysLT1R Ab. Concentration-depen- IL-4, two prototypic Th2 cytokines that share many biological effects, dent (B) and time-dependent (C) effects are presented as mean channel fluores- .(p Ͻ 0.01 vs untreated cells ,ءء ;p Ͻ 0.05 ,ء ;cence values (n ϭ 4Ð6 up-regulate the expression of the CysLT1R in human monocytes and monocyte-derived macrophages. This up-regulation involves tran- scriptional activation of the CysLT1R gene and is associated with enhanced CysLT1R protein expression and augmented responsive- (11, 25, 26). Its preferential role in asthma may be due, in part, to its ness to the LTD4 in terms of intracellular calcium mobilization longer half-life in vivo and its higher levels in asthmatic lung, com- and chemotactic activity. In contrast, the prototypic Th1 cytokine pared with IL-4, and, in part, to its predominant effect on some fea- ␥ IFN- did not modify CysLT1R expression. tures of asthma, such as goblet cell activation (11, 25). The pathology associated with asthma is thought to be mediated by Of the different mediators that are known to be involved in asthma, Th2 cells and involves potentially important roles for the cytokines LTs are considered to be among the most important because they IL-4, IL-13, and IL-5, because both mRNA and protein levels of these participate in both the bronchoconstriction and the inflammatory com- cytokines are elevated in allergic patients as compared with normal ponents of the disease. In general, cyclooxygenase metabolites of ara- individuals (7Ð10, 24). A particular association between IL-13 and chidonate are associated with nonallergic inflammation, whereas 5-li- asthma has also been suggested by several studies. IL-13 was pro- poxygenase metabolites such as LTB4, LTC4, and LTD4 are duced by BAL cells of atopic asthma patients after allergen challenge predominantly involved in allergic inflammation. In this context, it is (7), and increased IL-13 mRNA was detected in the bronchial mucosa quite interesting that the allergy-associated cytokines IL-13 and IL-4 of asthmatic patients (10). More recently, several studies have dem- tend to suppress cyclooxygenase-2 activity (17, 27), whereas they onstrated the central role IL-13 appears to play, at least in experimen- enhance LTA4 hydrolase activity, resulting in the increased produc- tal asthma, in terms of induction of AHR and mucus hypersecretion tion of LTB4 (28). In other reports, IL-13 was shown to enhance The Journal of Immunology 2859

2ϩ FIGURE 4. [Ca ]i was determined using the flu- orescent dye fura 2-AM. A, Human monocytes were cultured for 24 h in the absence or presence of IL-13 or IL-4 (10 ng/ml), loaded with fura 2-AM, and stim-

ulated with 100 nM LTD4 or 100 nM LTB4,inthe absence or presence of the CysLT1R antagonist MK571 (1 ␮M). B, Human monocyte-derived mac- rophages were cultured for 24 h in the absence or presence of IL-13 or IL-4 (10 ng/ml), loaded with

fura 2-AM, and stimulated with 100 nM LTD4.

15-hydroxyeicosatetraenoic acid release in monocytes (19, 29), to in- of the airways and other target organs are characteristic features of

crease cytosolic phospholipase A2 expression, and to modulate zy- asthma and allergic diseases. Thus, priming of leukocytes with cyto- mosan-stimulated arachidonic acid mobilization (30). kines such as IL-13 and IL-4, combined with an increase in CysLT Excessive production of leukocytes and their subsequent invasion production in the airways of asthmatics, may contribute to the influx

FIGURE 5. Effect of IL-13 and IL-4 on chemotactic response of monocytes to LTD4. A, Monocytes were incubated in the absence or presence of 10 ng/ml ␮ IL-13 or IL-4 for 24 h and then assessed for their ability to migrate across a 5- m-pore-size polycarbonate filter in response to graded concentrations of LTD4. Ͻ ءء Ͻ ء Results are from three independent experiments. , p 0.05; , p 0.01 vs untreated cells. B, Comparative responses of monocytes to LTD4 (100 nM) and MCP-1 (50 ng/ml) following incubation for 24 h in the presence or absence of 10 ng/ml IL-13 or IL-4. Chemokinesis was also assessed in the presence of vehicle

(M/M) or LTD4 (D4/D4) in both chambers and was not affected by pretreatment with the cytokines. 2860 IL-13 AND IL-4 INDUCE LEUKOTRIENE D4 RECEPTOR EXPRESSION and activation of leukocytes. In another receptor system, IL-13 and 10. Humbert, M., S. R. Durham, P. Kimmitt, N. Powell, B. Assoufi,R.Pfister, G. Menz, IL-4 were shown to augment the expression of the chemokine recep- A. B. Kay, and C. J. Corrigan. 1997. Elevated expression of messenger ribonucleic acid encoding IL-13 in the bronchial mucosa of atopic and nonatopic subjects with tors CXCR1 and CXCR2 in human monocytes (31). Hence, our find- asthma. J. Allergy Clin. Immunol. 99:657. ings, in addition to the latter observations, suggest that, in Th2-dom- 11. Grunig, G., M. Warnock, A. E. Wakil, R. Venkayya, F. Brombacher, D. M. Rennick, D. Sheppard, M. Mohrs, D. D. Donaldson, R. M. Locksley, and D. B. 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