IL-13 increases the cPLA2 gene and protein expression and the mobilization of arachidonic acid during an inflammatory process in mouse peritoneal macrophages

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IL-13 increases the cPLA2 gene and protein expression and the mobilization of arachidonic acid during an in£ammatory process in mouse peritoneal macrophages Astrid Rey a , Christine M'Rini a; b , Patricia Sozzani a , Yves Lamboeuf a , Maryse Beraud a , Daniel Caput c , Pascual Ferrara c , Bernard Pipy a; * a

Laboratoire Macrophages, Me¨diateurs de l'In£ammation et Interactions Cellulaires, UPS E.A. 2405, Baªtiment L1, Hoªpital de Rangueil, 31403 Toulouse cedex 4, France b Service de Physiologie, Faculte¨ de Me¨decine de Rangueil, 118 route de Narbonne, 31062 Toulouse, France c Sano¢ Recherche, P.O. Box 137, 31676 Labege Innopole, France Received 16 June 1998; accepted 26 June 1998

Abstract Pretreatment of mouse peritoneal macrophages with interleukin-13 (IL-13) potentiates the mobilization of arachidonic acid (AA) and the production of HETEs but does not affect the production of cyclooxygenase metabolites triggered by the suboptimal concentration of an inflammatory agonist (opsonized-zymosan). Cycloheximide suppresses these effects of IL-13 suggesting that de novo protein synthesis is involved. Indeed, IL-13 induces a time-dependent increase in the levels of cytolosic PLA2 (cPLA2) protein and mRNA. This study demonstrates a new pathway for IL-13 to modulate the inflammatory process in macrophages via modifications of cPLA2 expression and subsequent AA mobilization. ß 1998 Elsevier Science B.V. All rights reserved. Keywords: Arachidonic acid; Eicosanoid; Interleukin-13; Cytosolic phospholipase A2; Opsonized-zymosan; Mouse peritoneal macrophage

1. Introduction Macrophages are involved in immunological and non-immunological responses, such as phagocytosis, presentation of antigen or secretion of numerous different protidic and lipidic substances [1,2] acting as

Abbreviations: cPLA2, cytosolic phospholipase A2; AA, arachidonic acid; IL-13, interleukin-13 * Corresponding author. Fax: +33 5-61-32-22-93; E-mail: [email protected]

e¡ector or modulatory cells. Among the lipid mediators secreted by macrophages, prostaglandins and leukotrienes are known to have a strong involvement in symptomatology and regulation of in£ammation [3,4]. These bioactive lipids are produced by oxygenation of arachidonic acid (AA) by cyclooxygenase and lipoxygenase pathways. Before this fatty acid is oxygenated, it is released from cell membrane lipids by lipases, particularly by phospholipases A2 (PLA2) that speci¢cally hydrolyze the sn-2-ester bond of phospholipids, resulting in the production of free fatty acid and lysophospholipids. Three di¡erent types of PLA2 have been found in macrophages

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[5], a secretory PLA2 (sPLA2) [6], a cytosolic Ca2‡ independent PLA2 (iPLA2) [7] and a cytosolic PLA2 (cPLA2) [8,9]. Because of its preference for sn-2 AA [10,11], cPLA2 is considered to be the key enzyme in the synthesis of leukotrienes and prostaglandins. Consequently, the initial activation of cPLA2 represents a critical regulatory step in the control of in£ammatory responses mediated by leukotrienes and prostaglandins. Interactions between cytokines and macrophages are of great importance in the regulation of in£ammatory processes by inducing a wide spectrum of macrophage di¡erentiation states [1]. Interleukin-13 (IL-13), a Th2-type cytokine, has been described for its ability to change morphology and functions of macrophages [12]. This cytokine enhances MHC class II antigen and mannose receptor expression, down-modulates CD14 and FCRQ expression [13,14], inhibits nitric oxide [15], proin£ammatory cytokine [16] and monocyte chemoattractant protein-1 production [17], and, as shown in our laboratory, negatively modulates TPA-induced respiratory burst in human monocytes [18]. IL-13 is also able to block the in£ammatory cyclooxygenase pathway of LPS-stimulated monocytes [19] and to increase the expression and activity of 15-lipoxygenase which catalyzes the C-15 hydroperoxydation of AA, leading to the production of the anti-in£ammatory mediator 15-HETE [20]. These latter results show that IL-13 can interfere in the metabolism of AA. However, the e¡ect of this cytokine on cPLA2, the key enzyme of AA metabolism, is not yet known. In this study, we have examined the e¡ect of IL-13 on the AA metabolism of mouse peritoneal macrophages triggered by a potent phagocytic and in£ammatory stimulus, opsonized-zymosan (OZ). We have also studied the e¡ect of IL-13 on the cPLA2 protein and mRNA content of macrophages. We report here that IL-13 enhances macrophage [3 H]AA mobilization and [3 H]HETE production challenged by OZ, at suboptimal level of stimulation. Furthermore, we demonstrate that IL-13 induces a time-dependent increase in the levels of cPLA2 protein and mRNA in macrophages.

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2. Materials and methods 2.1. Materials The following were used: labeled [5,6,8,9,11,12, 14,15-3 H]arachidonic acid ([3 H]AA) spec. act. 200 Ci/mmol (Amersham Corp., France); Dulbecco's modi¢ed Eagle medium (DMEM) (Gibco, CergyPontoise, France); zymosan A (Sigma Chimie, La Verpillie©re, France). OZ was prepared by mixing one part of fetal calf serum with one part of boiled saline-washed zymosan for 30 min at 37³C. The zymosan particles were removed by centrifugation prior to the use of OZ. IL-13 was produced in transformed Chinese hamster ovary cells by Sano¢ (Sano¢ Recherche, Labege, France). All other chemicals and solvents were reagent grade. 2.2. Isolation and culture of macrophages Mouse peritoneal macrophages were harvested as described by Cohn and Benson [21] from female Swiss-OF1 mice (IFFA-CREDO, l'Arbresle, France). For AA metabolism analysis, macrophages were cultured into 24-well culture plates (106 cells/well) and labelled for 18 h with 1 WCi [3 H]AA in the presence (IL-13-treated cells) or absence (control cells) of IL13 (10 ng/ml) in DMEM containing 1% fetal calf serum (FCS). For cPLA2 analysis, macrophages were cultured into 6-well culture plates (5U106 cells/well) for 18 h and IL-13 was added to the culture medium (DMEM/1% FCS) at di¡erent times during this 18 h culture in order to avoid the culture-time e¡ect on cPLA2 expression. 2.3. Analysis of basal and in£ammatory-induced [3 H]AA metabolism in presence or absence of IL-13 To determine [3 H]AA incorporation in the presence of IL-13, some control and IL-13-treated wells were removed from the wells after the [3 H]AA incorporation period and the membrane phospholipids and neutral lipids were extracted and analyzed as previously described [22]. For determination of basal and in£ammatory-induced [3 H]AA metabolism, macrophages were washed at the end of the [3 H]AA incorporation period and incubated for 1 h in DMEM/

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1% FCS in the absence or in the presence of OZ at di¡erent doses (12.5^200 Wg/ml). At the end of this incubation period, medium was collected and the various [3 H]AA oxygenation products released by macrophages (cyclooxygenase and lipoxygenase products) were measured by thin-layer chromatography (TLC) [22]. Brie£y, supernatants were acidi¢ed to pH 5.4 with 1 M HCl and chromatographically extracted using Varian Bond Elut C18 columns (Analytichem). AA metabolites were eluted with methanol. The methanol samples were evaporated to dryness under nitrogen. The residues were dissolved in 80 Wl of methanol and applied to thin-layer silica gel plates (LK-6-DF Whatman), which had been previously activated (1 h at 100³C). The solvent system used for the separation of eicosanoids was the organic phase of ethyl acetate-water-isooctane-acetic acid (110:100:50:20, v/v). Samples were monitored by rapid scanning with the Berthold LB2821 TLC-linear analyzer.

and subjected to ampli¢cation. The polymerase chain reaction (PCR) was performed with primers constructed by Isoprim from published cDNA sequence of murine cPLA2 (5P-CAC TCA CCA AGG CCA TTA TCA T-3P and 5P-GAG CTG ATG TTT GCA GAT TGG-3P) and glyceraldehyde phosphate dehydrogenase (GAPDH) to demonstrate that equal quantities of mRNA were subjected to ampli¢cation (5P-TCC ATG ACA ACT TTG GCA TCG TGG-3P and 5P-GTT GCT GTT GAA GTC ACA GCA GAC-3P). After denaturation at 94³C for 2 min, ampli¢cation consisted of denaturation at 94³C for 1 min, primer annealing at 60³C for 1 min, and extension at 72³C for 1 min. The reaction was ended by chilling at 4³C. Each ampli¢ed DNA was separated by electrophoresis on a 3% agarose gel containing 2% ethidium bromide. Gels were photographed and the quanti¢cation of signals was performed by using a scanner employing the programs Adobe Photoshop and Morphostar (IMSTAR, Paris, France).

2.4. Analysis of macrophage cPLA2 protein level by Western blotting

2.6. Statistical analysis

After incubation with IL-13, macrophages were washed twice with ice-cold PBS and scraped o¡ in a lysis bu¡er containing 250 mM sucrose, 50 mM HEPES, 1 mM EDTA, 1 mM phenylmethylsulfonyl £uoride, 10 Wg/ml aprotinin and 10 Wg/ml leupeptin. The protein content was determined using the Bradford protein assay kit (Bio-Rad). Then 10 Wg of total protein was separated on a 7.5% SDS-PAGE and transferred onto a nitrocellulose membrane (BioRad, Richmond, CA). The membrane was incubated with an anti-cPLA2 monoclonal antibody (1/500) (Santa Cruz Biotechnology, France) for 1 h. Immune complexes were revealed by an anti-mouse IgG peroxidase-conjugated antibody (1/1000) and visualized by an ECL system. 2.5. Analysis of macrophage cPLA2 mRNA by RT-PCR Total macrophages RNA was extracted using `Extract All' kit (Eurobio), a modi¢cation of the guanidinium isothiocyanate procedure [23]. One microgram of RNA for each extraction was reverse transcribed by previously described methods [24]

Results are given as the mean þ standard error. The statistical signi¢cance of di¡erences between groups was analyzed by Student's t-test. 3. Results 3.1. E¡ect of IL-13 on basal and zymosan-stimulated AA metabolism of mouse peritoneal macrophages The presence of IL-13 into the incorporation medium of macrophages modi¢es neither the quantity of [3 H]AA incorporated in the membrane lipids (46.4 þ 2.6% of initial radioactivity in control macrophages vs. 52.4 þ 7.9% in IL-13-treated macrophages) nor the type of lipids in which the [3 H]AA has been incorporated. In control and IL-13-treated macrophages, AA was incorporated mainly into PC (39 þ 3 vs. 36.1 þ 0.5% of initial radioactivity), PS (6.3 þ 3.3 vs. 5.6 þ 0.8), PI/PE (18.3 þ 4.5 vs. 18.1 þ 4.1) and DAG (4.5 þ 0.4 vs. 5.6 þ 0.5). Following the [3 H]AA incorporation period cells were washed and incubated for a further hour in fresh culture medium. During this hour, control and IL13-treated macrophages released a similar basal level

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Fig. 1. Dose-dependent zymosan-stimulated release of 3 H radioactivity by mouse peritoneal macrophages. Mouse peritoneal macrophages were prelabelled for 18 h with [3 H]AA, washed and then incubated for 1 h with di¡erent concentrations of OZ. Total 3 H radioactivity released in the supernatant was determined as described in Section 2. Data are expressed as percent increase of the control value, i.e. those macrophages that were not stimulated by OZ. Values represent the mean þ standard error of three separate experiments, each performed in triplicate. The asterisks denote a signi¢cant di¡erence relative to the control value (*P 6 0.05; **P 6 0.01).

of 3 H radioactivity (4.5 þ 0.6 vs. 4.6 þ 1.3% of cellincorporated [3 H]AA). The study of the e¡ect of IL-13 on macrophage AA mobilization triggered by an in£ammatory agonist, was carried out by stimulating control and IL-13-treated with OZ during a further culture hour. Zymosan dose-response experiments indicate that OZ provokes an increase of the basal 3 H radioactivity released by control cells. As shown in Fig. 1, OZ induces a two-fold increase in 3 H radioactivity at 50 Wg/ml and the maximal stimulation is reached with the 100 Wg/ml concentration. Fig. 2A indicates that in IL-13-treated macrophages, the basal 3 H radioactivity released is also increased by OZ. Unlike in control cells, the maximal increase is obtained with 50 Wg/ml OZ instead of 100 Wg/ml. The 3 H radioactivity released by the cells is composed by free [3 H]AA and by 3 H-metabolites issued from the catabolism of this fatty acid by cyclooxygenase and lipoxygenase enzyme. As shown in Fig. 2, IL-13 induces a signi¢cant increase of free AA (Fig. 2B) and [3 H]HETEs (Fig. 2C) in macro-

Fig. 2. E¡ect of IL-13 on the zymosan-stimulated [3 H]AA metabolism of mouse peritoneal macrophages. Mouse peritoneal macrophages were prelabelled for 18 h with [3 H]AA, in the presence (hatched boxes) or absence (black boxes) of IL-13. Cells were washed and cultured for a further hour in the presence or absence of OZ. (A) Releases of total 3 H radioactivity. (B) Production of free [3 H]AA and sum of [3 H]cyclooxygenase and [3 H]lipoxygenase metabolites. (C) Production of each [3 H]eicosanoid stimulated by 50 Wg OZ. 3 H radioactivity was determined as described in Section 2. Data are expressed as percent increase of the control values, i.e. those obtained with macrophages not incubated with IL-13 and not stimulated by OZ. Values represent the mean þ standard error of three separate experiments, each performed in triplicate. The asterisks denote a signi¢cant di¡erence between zymosan-stimulated macrophages treated or not by IL-13 (*P 6 0.05).

phages stimulated by 50 Wg/ml OZ, but it has no e¡ect on [3 H]cyclooxygenase metabolites. Furthermore, IL-13 modi¢es neither the production of

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[3 H]cyclooxygenase nor the production of [3 H]lipoxygenase metabolites in macrophages stimulated by 100 Wg/ml OZ. 3.2. E¡ect of IL-13 on cPLA2 protein expression Since IL-13 modi¢es AA mobilization, stimulated by OZ, and as cPLA2 is known as the primary enzyme regulating AA metabolism in macrophages, a Western blot analysis was performed to determine whether IL-13 is able to modify cPLA2 expression in mouse peritoneal macrophages. Kinetic study demonstrates that IL-13 induces a gradual increase in cPLA2 protein which began at 2 h and continued up to 18 h (Fig. 3). Densitometry of the immunoblot quantitatively analyzed the IL-13 increase in cPLA2 protein as being 1.5-fold after 4 h and 2.5-fold after 18 h, compared with the control level (time of IL-13 treatment: 0 h). 3.3. E¡ect of cycloheximide on the IL-13-induced increase in cPLA2 expression and on the zymosan-stimulated AA mobilization To determine whether the IL-13-induced enhancement of AA mobilization, triggered by OZ, requires a de novo protein synthesis, parallel experiments were performed in the presence of cycloheximide. Our results show that cycloheximide partly prevents

Fig. 3. Time course of cPLA2 protein induction by IL-13 in mouse peritoneal macrophages. Macrophages were cultured 18 h in DMEM/1% FCS. The IL-13 stimulation was conducted during this 18 h incubation period by adding 10 ng/ml IL-13 for the aforementioned length of time (from 2 h to 18 h). Control macrophages were incubated for 18 h in DMEM/1% FCS without stimulation by IL-13 (time of IL-13 treatment: 0 h). cPLA2 protein expression was determined by Western blot analysis as described in Section 2.

Fig. 4. E¡ect of cycloheximide on IL-13-induced increase of cPLA2 protein level and of zymosan-stimulated total 3 H radioactivity in mouse peritoneal macrophages. After adhesion, some macrophages were used for cPLA2 protein analysis whilst others were used for eicosanoid analysis. (A) For cPLA2 analysis, macrophages were cultured for 18 h with (IL-13) or without (control) IL-13 (10 ng/ml) in the presence or absence of 8 WM cycloheximide (CHX). cPLA2 expression was then determined by Western blot analysis as described in Section 2. (B) For AA mobilization analysis, macrophages were cultured with or without IL-13 (10 ng/ml), in the presence or absence of 8 WM CHX during the 18 h labelling period. Cells were then washed and incubated for a further hour in the presence of OZ (50 Wg/ml). Total 3 H radioactivity released is expressed as percent increase of the control values, i.e. values obtained with macrophages not incubated with IL-13 and not stimulated by OZ. Values represent the mean þ standard error of three separate experiments, each performed in triplicate. The asterisks denote a signi¢cant di¡erence between the CHX- plus IL-13 and OZ-treated macrophages and the macrophages treated only with IL-13 and OZ (**P 6 0.01).

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3.4. E¡ect of IL-13 on cPLA2 mRNA content To determine whether the IL-13-induced increase of cPLA2 protein expression results from the modi¢cation of gene expression, the cPLA2 mRNA content was analyzed by RT-PCR in IL-13-treated cells. As shown in Fig. 5, IL-13 induces a time-dependent increase of the cPLA2 mRNA content in mouse peritoneal macrophages. The maximum increase is reached after 2 h incubation (a 20-fold increase of the mRNA content is observed compared with the control level), remains until 4 h and gradually decreases so that at 18 h there is only a 5-fold increase between the mRNA level of IL-13-treated and control macrophages (time of IL-13 treatment: 0 h). 4. Discussion

Fig. 5. Time course of cPLA2 mRNA induction by IL-13 in mouse peritoneal macrophages. Macrophages were cultured 18 h in DMEM/1% FCS. The IL-13 stimulation was conducted during this 18 h incubation period by adding 10 ng/ml IL-13 for the aforementioned length of time (from 2 h to 18 h). Control macrophages were incubated for 18 h in DMEM/1% FCS without IL-13 stimulation (time of IL-13 treatment: 0 h). At the end of the 18 h incubation period, total RNA was subjected to RT-PCR as described in Section 2. (A) Data shown are the ampli¢cation products of cPLA2 and GAPDH. (B) Densitometric values of cPLA2 mRNA expression after normalization for changes in the quantity of GAPDH transcript.

both the IL-13-induced increase of cPLA2 protein expression (Fig. 4A) and the IL-13-enhanced AA metabolism in zymosan-stimulated macrophages (Fig. 4B). In this experiments, densitometry of the immunoblot indicated that IL-13 induces a 7-fold increase in cPLA2 protein content compared to control cells, whereas only a 2.5-fold increase was observed in the presence of CHX.

Previous reports describing the e¡ects of IL-13 on ligand-stimulated AA metabolite production have focused on the cyclooxygenase and lipoxygenase enzymes involved in eicosanoid production [19,25]. None of them, however, has studied the key enzyme of AA metabolism: cPLA2. In the present study, we have examined the e¡ect of IL-13 on the AA metabolism of mouse peritoneal macrophages triggered by an in£ammatory agonist, opsonized-zymosan (OZ). We demonstrate that IL-13 modi¢es not only the zymosan-stimulated AA mobilization of macrophages but also the cPLA2 mRNA and protein content of macrophages. As already shown [26], macrophages labelled for several hours by [3 H]AA and then washed and cultured for a further hour spontaneously release 3 H radioactivity into their culture medium. The presence of this radioactivity due to [3 H]AA mobilization from macrophage lipid pools is composed of free [3 H]AA and 3 H-oxygenated metabolites originating from the catabolism of AA by lipoxygenases and cyclooxygenases. We show in our study that IL-13 modi¢es neither the spontaneous basal AA mobilization of [3 H]AA nor the ratio of the production of leukotrienes and prostaglandins, suggesting that this cytokine, for longer periods up to 18 h, has no role in the functioning of macrophage AA metabolism in basal conditions. Stimulation of macrophages by OZ provokes, as

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expected [27], a dose-dependent increase of the [3 H]AA mobilization. In IL-13-treated cells, the basal [3 H]AA mobilization and catabolism are also increased by OZ. However, there are qualitative and quantitative di¡erences in [3 H]AA metabolism of control and of IL-13-treated cells. Firstly, the [3 H]AA mobilization triggered by 50 Wg/ml OZ, suboptimal concentration, is enhanced by the IL-13 pretreatment and leads to a greater release of free [3 H]AA by macrophages. The physiological signi¢cance of this enhanced liberation of free AA remains to be established. In recent years, studies have proposed that AA itself may act as a second messenger in di¡erent cellular responses [28,29]. This free fatty acid has been shown to modulate the induction of proin£ammatory genes [30,31], and to interact with proteins involved in signalling cascades such as tyrosine kinase [32], PKC [33] or Rho [34]. This free fatty acid can also induce cellular responses which are independent of its action on PKC such as activation of calcium extrusion in macrophages via action on a calcium ATPase [35]. So, the role of the free AA on the IL-13-induced responses of macrophages must be considered. The second di¡erence between the [3 H]AA metabolism of control and IL-13-treated cells, after OZ stimulation, is that the IL-13-enhanced [3 H]AA mobilization leads to a signi¢cantly higher production of [3 H]HETEs, while the production of LTC4 -D4 , LTB4 , and cyclooxygenase metabolites is not modi¢ed. These ¢ndings suggest that the IL-13 pretreatment of cells had no e¡ect on the enzymes, namely 5lipoxygenase and cyclooxygenases, implicated in the production of these metabolites. This suggestion is in agreement with recent works of Nassar et al. showing that IL-13 does not modify the 5-lipoxygenase expression in monocytes whereas it enhances the production of 15-HETEs and the expression of 15-lipoxygenase [25]. Endo et al. have demonstrated that IL-13 is able to suppress LPS-induced PGE2 release in human monocytes by downregulating the cyclooxygenase-2 protein and mRNA expression [19]. These data and our present data show that IL-13 can act on the functioning of macrophage AA metabolism but only in speci¢c conditions such as those in our study, during an in£ammatory process. This reinforces the idea that IL-13 has a modulatory rather than a real stimulating role. Furthermore,

the augmentation of macrophage AA mobilization stimulated by a suboptimal concentration of OZ and the lack of e¡ect of IL-13 on cells stimulated by an optimal concentration indicate that the ability of this in£ammatory agonist to stimulate the AA metabolism is modulated by the IL-13-induced activation state of macrophages. Thus, when the optimal response triggered by the stimulus is reached, IL-13 fails to modify the AA mobilization of macrophages. Besides, the fact that the IL-13 pretreatment does not modify basal AA mobilization, whereas it does modulate the zymosan-stimulated one, allows us to speculate on a reason for this di¡erence. In mouse peritoneal macrophages, zymosan has been shown to increase the levels of cPLA2 phosphorylation resulting from AA release [36]. The cPLA2 requires phosphorylation, translocation to membrane and calcium for activity [37] So, IL-13 treatment may not be enough to elicit the full enzyme activity without other stimulus intervention. We also contemplated whether or not IL-13 is able to modify the cPLA2 protein expression. Our studies demonstrated that (i) IL-13 induces a time-dependent increase in the amount of cPLA2 protein and that (ii) the addition of cycloheximide, a translational inhibitor, during IL-13 treatment suppresses the cytokine e¡ects on both AA mobilization and protein synthesis. Therefore, considering these results, we hypothesize that the IL-13 e¡ect on AA mobilization is the consequence of the cytokine e¡ect on cPLA2 synthesis. To test this hypothesis, the cPLA2 mRNA content of IL-13-treated macrophages were studied using RT-PCR analysis. We demonstrated that the level of cPLA2 protein continued to increase between 4 h and 18 h, whereas the level of cPLA2 mRNA level decreased. This di¡erence between the time course of cPLA2 mRNA and protein levels could be explained by a relatively long half-life of cPLA2 protein in the IL-13-treated cells. Our results indicate that IL-13 modi¢es the cPLA2 expression at the transcriptional and translational level, and suggest that the cytokine may modi¢es the post-translational regulation of cPLA2. The regulation of cPLA2 synthesis by cytokines has to date been studied particularly for proin£ammatory cytokines, such as IL-1 and INFQ. Unlike IL13, cPLA2 protein synthesis induced by this cytokine is correlated with enzyme activity and with PGE2

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production without stimulus intervention [38,39]. Consequently, even if IL-13 has the same e¡ect as proin£ammatory cytokines on cPLA2 synthesis, its e¡ect on AA metabolism di¡ers totally as IL-13 mainly induces the release of AA rather than that of in£ammatory mediators. Thus, the remaining question concerns the role of this AA at the in£ammatory sites: does it act directly as an intracellular messenger or indirectly via its transcellular metabolism pathway? In conclusion, our study reports for the ¢rst time that the induction of cPLA2 expression by a Th2type cytokine reinforces the modulatory role of IL13 on the in£ammatory response. The IL-13-induced change in the production of free AA and HETEs may add to the capacity of IL-13 to inhibit the secretion of proin£ammatory cytokines to limit the development of in£ammation. Thus, IL-13, according to its e¡ects on the functions of macrophages, appears to be able to prevent cell damage while still promoting repair by allowing macrophages to participate in the immune response. Acknowledgements We wish to thank Mrs. M.F. Frisach for her assistance in the accomplishment of this work.

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