©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Interleukin-13 Inhibits Protein Kinase C-triggered Respiratory Burst in Human Monocytes
ROLE OF CALCIUM AND CYCLIC AMP (*)

(Received for publication, October 3, 1994; and in revised form, December 9, 1994)

Patricia Sozzani (1) Claudie Cambon (1)(§) Natalio Vita (2) Marie-Hélène Séguélas (1) Daniel Caput (2) Pascual Ferrara (2) Bernard Pipy (1)

From the  (1)From INSERM CJF 9107, IFR L. Bugnard, Université P. Sabatier, CHU Rangueil, 31054 Toulouse Cedex, France and (2)Sanofi Recherche, BP 137, 31676 Labège Innopole, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Interleukin-13 (IL-13), a novel cytokine produced by activated lymphocytes modulates some monocyte functions, but no data is available concerning the signal transduction pathway. We show here, the inhibitory effect of IL-13 on 12-O-tetradecanoylphorbol-13-acetate (TPA)-triggered reactive oxygen intermediate production in human monocytes and the signals involved in this response. Our results show that IL-13 produces rapid and transient phosphoinositide hydrolysis and intracellular Ca mobilization. Furthermore, IL-13 induces intracellular cAMP accumulation through inositol 1,4,5-trisphosphate-dependent Ca mobilization. Metabolic inhibitors were used to relate the first steps in signaling pathways to the inhibitory effect of IL-13 on TPA-triggered reactive oxygen intermediate production. Indeed, inhibitors of phospholipase C (neomycin), intracellular Ca mobilization (8-[N,N-diethylamino]-octyl 3,4,5-trimethoxybenzoate hydrochloride), adenylate cyclase (Delta^9-tetrahydrocannabinol), and protein kinase A (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide) impair the IL-13 inhibitory response. Altogether these observations indicate that modulatory effect of IL-13 on the TPA-induced oxidative burst is the result of the intracellular cAMP accumulation through an inositol 1,4,5-trisphosphate-induced Ca mobilization-dependent pathway.


INTRODUCTION

Interleukin 13 (IL-13) (^1)is a novel lymphokine of 112 amino acids with a molecular mass of 12.5 kDa produced by activated Th-2 cells(1) . This cytokine induces cell surface phenotype changes and displays immunomodulatory effects on B cells and human monocytes ( (2) and for reviewed, see (3) ). In B cells, IL-13 induces proliferation and differentiation(4) , promotes CD23 expression and the production of certain immunoglobulin isotypes such as IgG4 and IgE(5) . IL-13 displays many effects on monocytes. It induces morphological changes (2) and enhances the expression of several members of the integrin superfamily and class II major histocompatibility complex antigens, whereas it down-regulates the expression of CD14 and FcR receptors (CD64, CD32, and CD16)(6) . IL-13 inhibits immunodeficiency virus type 1 production, but the precise mechanism remains to be elucidated(7, 8) . It reduces pyrogen-induced expression of procoagulant activity(9) . In lipopolysaccharide-stimulated monocytes(1, 6) , IL-13 inhibits the production of chemokines (IL-8, macrophage inflammatory proteins 1alpha), hematopoietic growth factors (granulocyte/macrophage colony-stimulating factor, granulocyte colony-stimulating factor), and proinflammatory cytokines (e.g. tumor necrosis factor alpha, IL-1, IL-6). Thus, IL-13 is considered as an anti-inflammatory cytokine.

However, the effect of IL-13 on the reactive oxygen intermediates (ROI) production, an important function of monocytes that are involved in infectious and inflammatory processes, has not yet been investigated. The NADPH-oxidase system is the key step in the regulation of ROI generation. The oxidase is normally dormant in resting phagocytes, but it can be rapidly activated by a number of stimuli such as protein kinase C agonists like phorbol esters, 12-O-tetradecanoylphorbol-13-acetate (TPA). It is known that the oxidase requires interaction at the plasma membrane level between membrane and cytosolic components, these include flavocytochrome b, the cytosolic proteins p47 and p67, and the small GTP-binding proteins Rap1A, Rac-1, and Rac-2 (for review, see (10, 11, 12) ). Classically, protein kinase C phosphorylates specific proteins (p47, p67) that constitute the predominant regulatory mechanism governing the activation of the oxidase(13) .

While several studies have been oriented toward phosphorylation and activation of respiratory burst, the reversal of the event by dephosphorylation and inactivation of the active phagocyte to the normal resting state have not been fully elucidated. Agents that increase the intracellular concentration of cAMP have been shown to be negative modulators of TPA-triggered respiratory burst in polymorphonuclear leukocytes (14) and in Ehrlich ascites tumor cells (15) . The regulation of intracellular cAMP level involves at least two independent systems, a G protein pathway coupled with adenylate cyclase and the direct activation of catalytic subunits of adenylate cyclase. In particular, the Ca-calmodulin complex can activate specific I and III adenylate cyclase isozymes(16) . A coupling of phospholipase C and adenylate cyclase effector systems through Ca has also been reported in leukocytes(17, 18) .

In this study, we investigate the capacity of IL-13 to modulate respiratory burst in human monocytes and the signaling pathways used by the cytokine to modify this function. Our findings indicate that IL-13 inhibits TPA-triggered ROI production by a mechanism involving a rapid and transient phospholipase C-dependent Ca mobilization and the consequent protein kinase A activation.


EXPERIMENTAL PROCEDURES

Materials

Purified recombinant human IL-13 (IL-13) was produced in transformed Chinese hamster ovary cells and purified as described before(1) . As optimal responses were obtained with 100 ng/ml, this concentration of IL-13 was used in all the experiments described. The lipopolysaccharide concentration (measured by Limulusamoebocyte lysate assay) was always less than 30 pg/µg of protein. The neutralizing anti-IL-13 antiserum was obtained in rabbits using the purified recombinant cytokine as immunogen. Briefly, 50 µg of IL-13 dissolved in 0.5 ml of saline and emulsified with an equal volume of Freund's complete adjuvant were injected subcutaneously. Two, three, and four weeks after the first immunization, the same amount of IL-13 was injected in Freund's incomplete adjuvant. Rabbits were bled at the same time, and the titers of the antisera were followed by radioimmunoassay as described before (19) using an iodinated IL-13 as tracer. (^2)Special for macrophages medium, RPMI 1640, inositol-free RPMI 1640, fetal calf serum, and Hanks' balanced salt solution with or without Ca were from Life Technologies, Inc. myo-[2-^3H]Inositol, D-myo-[2-^3H]inositol 1-phosphate, D-myo-[2-^3H]inositol 1,4-bisphosphate, D-myo-[2-^3H]inositol 1,4,5-trisphosphate, D-myo-[2-^3H]inositol 1,3,4,5-tetrakisphosphate, and complete phase-combining system liquid scintillant were from Amersham Corp. 12-O-Tetradecanoylphorbol-13-acetate (TPA), 8-[N,N-diethylamino]-octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB8), forskolin, N^6,2`-O-dibutyryladenosine 3`:5`-cyclic monophosphate (dibutyryl cAMP), 1-isobutyl-3-methylxanthine, Delta^9-tetrahydrocannabinol (Delta^9-THC), neomycin sulfate, and indomethacin were from Sigma, and Fluo 3-acetoxymethylester (Fluo 3-AM) was from Molecular Probes (Eugene, OR). N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89) was from France Biochem, Meudon, France. All other chemicals and solvents were reagent grade.

Cell Isolation

Peripheral blood mononuclear cells were separated by a standard Ficoll-Hypaque (MSL, Eurobio, Les Ullis, France) gradient method. Human monocytes were isolated from human peripheral blood mononuclear cells by adherence to plastic for 2 h in special for macrophages medium, supplemented with 5% fetal calf serum, 2 mML-glutamine, and antibiotics, at 37 °C in a humidified atmosphere containing 5% CO(2). Nonadherent cells were removed by washing, and remaining adherent cells (monocytes purity was >95%) were cultured overnight. They were designated as monocytes according to CD14 antigen expression and nonspecific esterase staining(20) .

Assay of ROI Production

Peripheral blood mononuclear cells (6 times 10^5/assay) were seeded into luminometer sterile cuvettes, and ROI production was measured by chemiluminescence (CL) in the presence of luminol (60 µM), using a thermostatically (37 °C) controlled luminometer 1251 LKB as described previously (21) . The generation of CL in human monocytes triggered with 100 nM TPA (time 0) was continuously monitored for 10 min, and the area under the curve (expressed in mV/s) was statistically analyzed using a Hewlett Packard 85 computer. Experiments were performed in Hanks' balanced salt solution medium with or without Ca. In some experiments, cells were incubated for 10 min before TPA in the presence of metabolic inhibitors such as Delta^9-THC (1 µM), an inhibitor of adenylate cyclase, H89 (10 µM), an inhibitor of protein kinase A, neomycin sulfate (10 µM), an inhibitor of phospholipase C, TMB8 (10 µM), an inhibitor of InsPinduced calcium release from intracytoplasmic stores, and indomethacin (0.5 µM), an inhibitor of the cyclooxygenase pathway. In all cases IL-13 or vehicle (Hanks' balanced salt solution) was added 5 min before TPA. In parallel experiments, IL-13/anti-IL-13 antiserum complex was used alone at time 0 or added 5 min before TPA.

Measurements of [^3H]Inositol Phosphates

Peripheral blood mononuclear cells (4 times 10^6/well) were seeded into 24-well culture plates. Adherent monocytes were incubated in RPMI 1640 inositol-free medium containing 0.04% bovine serum albumin in the presence of 7 µCi of myo-[2-^3H]inositol/well, for 24 h at 37 °C. The cells were washed 3 times and then stimulated by IL-13. The reaction was stopped at different times up to 5 min by the addition of 1 ml of ice-cold methanol. Extraction of [^3H]inositol phosphates was performed as described elsewhere(22) . Briefly, 2 ml of chloroform and 0.3 ml of water were added to the methanol-treated cells in order to obtain a final chloroform/methanol/water ratio of 2:1:0.6. The top aqueous layer was collected, and [^3H]inositol phosphates were reextracted from the chloroform/methanol phases using 0.6 ml of water. The inositol phosphates were separated using anion exchange SAX Amprep minicolumn (Amersham Corp.) and, successively eluted with ammonium formate, 0.1 M formic acid using ammonium formate increments from 0.1 to 1.2 M. In our conditions, [^3H]InsP standards, InsP(1), InsP(2), InsP(3), and InsP(4) were eluted at 0.1, 0.4, 0.8, and 1.2 M ammonium formate, 0.1 M formic acid, respectively. Total eluate volume was mixed with complete phase-combining system scintillant, and radioactivity was measured using a liquid scintillation counter.

Determination of Intracellular Calcium Concentration

Intracellular calcium concentration was measured in single cells by a video digital microscopy technique using the fluorescent probe Fluo 3-AM. Briefly, peripheral blood mononuclear cells (2 times 10^6 cells) were plated into 60-mm diameter plastic culture dishes, and adherent monocyte monolayers were loaded with 15 µM Fluo 3-AM for 1 h at 37 °C(23) . The cells were then washed 5 times with Hanks' balanced salt solution (with or without Ca). The time course of the intracytosolic Ca level was recorded every 2 s for a total period of 2 min after the addition of IL-13. In parallel assays, cells were preincubated with 10 µM TMB8 for 10 min before the addition of IL-13. Ionomycine (2.5 µM) was added at the end of each experiment to assess the maximum intensity response. Cells were visualized with an inverted microscope (Nikon Diaphot 300). The light source was a xenon lamp XBO 100 watts (Osram, Munich, Germany). Excitation (488 nm) and emission (525 nm) wavelengths were selected by a XF23 filter block (Nikon). They were acquired by an intensified camera LHESA, LH 5038-STD (Cergy Pontoise, France). Images were digitized, and the fluorescence was analyzed using the Imstar starwise/fluo software system (Paris, France).

Measurements of cAMP Accumulation in Human Monocytes

Peripheral blood mononuclear cells (4 times 10^6/well) were seeded into 24-well culture plates. Adherent cells were washed twice with incubation buffer (RPMI 1640, 1 mg/ml bovine serum albumin, 0.5 mM 1-isobutyl-3-methylxanthine) and incubated at room temperature with IL-13 for different periods of time (ranging from 1 to 15 min). Forskolin (10 µM) was used as positive control. To stop the reaction, the medium was discarded, and the cells were lysed with 95% cold ethanol. After an incubation of 5 min at 0 °C, the lysate was removed, lyophilized, and finally dissolved in radioimmunoassay buffer(24) . cAMP was quantified in triplicate using a cAMP I-labeled radioimmunoassay system from Amersham Corp. In parallel experiments, the kinetics of cAMP production were followed in cells preincubated for 10 min with neomycin sulfate (0.5 mM) or TMB8 (10 µM) before IL-13 addition.

Statistical Analysis

Results are expressed as mean ± S.E. Data were analyzed by one-way analysis of variance, and the multiple comparisons of each treatment were calculated applying the Tukey method(25) .


RESULTS

IL-13 Inhibits ROI Production Induced by TPA

TPA induces ROI production as measured by continuous CL kinetics generation. IL-13 had no effect on the basal oxidative response of unstimulated monocytes, whereas, when added 5 min before the protein kinase C agonist, the cytokine produced an inhibition of TPA-triggered ROI production (Fig. 1). The total CL emission, area under curve (Fig. 2), and CL maximum (data not shown) were both 35% lower compared with the TPA control. Moreover, we found that IL-13 antibody completely reversed the IL-13 inhibitory effect on oxidative burst. IL-13/anti-IL-13 antiserum complex per se was not able to induce ROI production (Fig. 1).


Figure 1: Effect of IL-13 pretreatment on TPA-triggered oxidative burst in human monocytes. The generation of CL was monitored after TPA (100 nM) (a), IL-13 (100 ng/ml) (d), Hanks' balanced solution (control) (e), or IL-13/anti-IL-13 antiserum complex (f) addition at time 0, as described under ``Experimental Procedures.'' In parallel experiments, monocytes were incubated for 5 min before TPA with IL-13 (100 ng/ml) (c) or IL-13/anti-IL-13 antiserum complex (b). The curves are representative of three separate experiments, each performed in triplicate.




Figure 2: Effect of IL-13 pretreatment on TPA-triggered chemiluminescence production in human monocytes in the absence (0Ca) or in the presence of extracellular calcium and metabolic inhibitors. Total CL emission (area under curve) was observed for 10 min after TPA (100 nM) (&cjs2108;) or IL-13 (100 ng/ml)+TPA (&cjs2113;) stimulation with or without (CONTROL) inhibitors as described under ``Experimental Procedures.'' When inhibitors were used, monocytes were preincubated (10 min) with 1 µMDelta^9-THC, 10 µMH89, 10 µM neomycin sulfate (NEO), 10 µMTMB8, 0.5 µM indomethacin (IND). Data are means ± S.E. of three separate experiments, each performed in triplicate.**, p < 0.01.



To investigate the possible involvement of the cAMP-dependent protein kinase pathway in the inhibitory effect of IL-13 on TPA-triggered ROI production, cells were preincubated with Delta^9-THC, an inhibitor of adenylate cyclase, or H89, an inhibitor of protein kinase A. The results obtained show that these inhibitors completely block the effect of IL-13 (Fig. 2). Moreover, forskolin (10 µM), a direct activator of adenylate cyclase, or dibutyryl cAMP (5 mM), a cell permeable analogue of cAMP, caused 50% inhibition of ROI production induced by TPA. These effects were reversed in the presence of H89 (10 µM).

We also investigated Ca mobilization as another candidate pathway in IL-13 modulation of the oxidative burst. In the absence of extracellular Ca, the effect of IL-13 on TPA-induced ROI production was maintained. In the presence of neomycin sulfate (inhibitor of phospholipase C) or TMB8 (inhibitor of InsP(3)-induced intracellular Ca release from intracysternal stores), the IL-13 inhibitory effect was impaired, suggesting that phospholipase C and subsequent production of InsP(3)-induced intracellular Ca release can be related to the inhibitory response (Fig. 2). The involvement of prostaglandin in this mechanism is not suggested. Indeed, in the presence of indomethacin (inhibitor of the cyclooxygenase pathway), the effect of IL-13 on TPA-triggered ROI production was maintained.

IL-13 Induces the Production of cAMP

Since cAMP seems to be involved in the inhibitory effect of IL-13 on TPA-induced ROI production, we measured the time course of cAMP accumulation induced by IL-13. Results show a rapid (1 min) and significant increase for 15 min (maximum time of experimental observation) (Fig. 3). In parallel, forskolin (10 µM) produced a similar kinetic of cAMP accumulation (data not shown). In order to study whether cAMP accumulation is linked to phospholipase C activation, neomycin sulfate and TMB8 were tested. Both inhibitors were able to block cAMP accumulation induced by IL-13, suggesting that phospholipase C activation and subsequent intracellular calcium release are necessary for the pathway leading to cAMP accumulation (Fig. 3).


Figure 3: Time course of cAMP accumulation induced by IL-13 in human monocytes. Monocytes were incubated with () or without (box) IL-13 (100 ng/ml). In parallel experiments, cells were preincubated for 10 min with (circle) neomycin sulfate (0.5 mM) or () TMB8 (10 µM) before IL-13 addition. cAMP was quantified as described under ``Experimental Procedures.'' Data are means ± S.E. of three separate experiments, each performed in triplicate. *, p < 0.05;**, p < 0.01.



Production of Inositol Phosphates by IL-13

The finding that the effect of IL-13 on ROI and cAMP production was reversed by neomycin suggests the involvement of phosphoinositide hydrolysis by phospholipase C. The results shown in Fig. 4confirm this event. Indeed, a rapid (15 s for InsP(2), InsP(3), and InsP(4), 30 s for InsP(1)), transient, and significant increase of all four inositol phosphate metabolites was observed when adherent monocytes prelabeled with myo-[2-^3H]inositol were incubated in the presence of IL-13.


Figure 4: Time course of IL-13-stimulated inositol phosphate formation in human monocytes. Monocytes were labeled with myo-[2-^3H]inositol, washed, and incubated with (bullet) or without (box) IL-13 (100 ng/ml). [^3H]inositol phosphates were measured as described under ``Experimental Procedures.'' Data are means ± S.E. of three separate experiments, each performed in triplicate. *, p < 0.05;**, p < 0.01.



IL-13 Induces Intracellular Ca Mobilization

At the single-cell level, a significant increase of the fluorescent Ca signal was observed within 30 s after the addition of IL-13 (Fig. 5A). In the absence of extracellular Ca, the IL-13-induced Ca response was maintained (Fig. 5B), suggesting that this increase is essentially a consequence of Ca release from intracysternal stores. To test this hypothesis, similar experiments were performed in the presence of TMB8. In these conditions, the increase of intracytosolic Ca induced by IL-13 was completely reversed (Fig. 5C).


Figure 5: Kinetics of [Ca] response in single cells after stimulation with IL-13. Monocytes were loaded with Fluo 3-AM, and, at the time indicated by arrows, IL-13 (100 ng/ml) was added to cells in Hanks' balanced solution with (A) or without (B) Ca. In parallel experiments, cells were preincubated for 10 min in the presence of 10 µM TMB8 (C) before IL-13 addition. Determination of [Ca] was measured as described under ``Experimental Procedures.'' Records of cell responses are representative of three separate experiments each performed in triplicate.




DISCUSSION

In the present study we determine the initial events induced by IL-13, which can regulate the protein kinase C-triggered respiratory burst in human monocytes. Classically, the activation of protein kinase C by TPA results in the activation of NADPH-oxidase, inducing the so-called respiratory burst(26, 27, 28) . Probably, phosphorylation of cytosolic oxidase proteins, p47 and p67 by activated protein kinase C is a key step in this response(11, 13) .

ROI and proinflammatory cytokines are likely to be involved in phagocyte antimicrobial activity and inflammatory responses. IL-13 produced by activated Th-2 cells is described to suppress the production of proinflammatory monokines(1, 6) . However, no information is available on the action of IL-13 on human ROI production.

Our results demonstrate that IL-13 inhibits the TPA-induced oxidative burst in human monocytes when added 5 min before TPA and also for longer periods up to 18 h (data not shown). The nonspecific inhibition of ROI production is excluded since in the presence of anti-IL-13 antiserum the effect of IL-13 is cancelled. The IL-13 down-modulatory effect on human monocyte oxidative burst supports that described with another anti-inflammatory cytokine, IL-4. Indeed, preexposure of human monocytes to IL-4 for 24 h decreases the respiratory burst induced by TPA(29) . IL-4 also inhibits this response when other stimuli such as zymosan, platelet-activating factor, chemotactic peptide and IFN- are used(29, 30) . An explanation for the similar biological activities displayed by IL-13 and IL-4 is that their receptors share a common subunit critical for cellular signal transduction(31) .^2 In contrast to the finding with human monocytes, the respiratory burst induced by TPA in murine resident peritoneal macrophages is enhanced by murine IL-4 used under similar experimental conditions (32) . Recent data show that P600 (murine IL-13) and murine IL-4 are unable to modify ROI response triggered by TPA in murine biogel-elicited macrophages(33) . These results suggest the necessity to be cautious when extrapolating data obtained with murine cells to the human system. As suggested for IL-4, the role of IL-13 in host defense and inflammation can be significant. ROI may damage various cells at high concentrations; however, their role is important in antimicrobial activity. The partial inhibition by IL-13 of TPA-triggered ROI production (35%) could reduce inflammatory reactions without completely abolishing the antimicrobial process.

Having established that IL-13 induces an inhibitory effect on ROI production, we investigated the signal transduction pathways used by this cytokine to induce the deactivation of the ROI response. In leukocytes, agents which increase intracellular cAMP concentrations (prostaglandins E(2) and E(1), dibutyryl cAMP, forskolin, and 1-isobutyl-3-methylxanthine) have often been shown to inhibit various cellular functions such as respiratory burst(34, 35, 36) . Some authors have recently proposed a mechanism of protein kinase A-dependent phosphorylation and subsequent activation of a specific phosphatase. This event results in dephosphorylation of NADPH-oxidase subunits and thus inactivation of the enzyme(14, 15) . Our data suggest the involvement of the cAMP-dependent protein kinase pathway in IL-13-induced inhibition of respiratory burst. Indeed, the direct involvement of cAMP in the IL-13 signaling pathway is demonstrated by a sustained increase of cAMP levels after cytokine stimulation. Furthermore, in the presence of adenylate cyclase (Delta^9-THC) and protein kinase A (H89) inhibitors, the effect of IL-13 on ROI production is not observed. IL-13 seems to mimic the forskolin or dibutyryl cAMP inhibitory effect on TPA-triggered ROI production.

Different studies show that the accumulation of cAMP can be subsequent to intracellular Ca mobilization(17, 37, 38) . Coupling of phospholipase C and cAMP pathways through Ca was reported in leukocytes (18, 39) and human neuroblastoma cells(40) . A direct activation of type I and III adenylate cyclase by Ca and calmodulin previously described (41, 42) could be a possible mechanism. Our experiments show that cAMP production is markedly reduced by prior treatment of human monocytes with an inhibitor of phospholipase C (neomycin sulfate) or an inhibitor of InsP(3)-induced Ca release (TMB8). These observations suggest that IL-13 causes intracellular cAMP accumulation by Ca-dependent processes.

It was of interest to verify the capacity of IL-13 to promote phospholipase C activation and intracellular Ca rise. We demonstrate here that IL-13 induces immediate and transient hydrolysis of inositol phospholipids and a rapid increase in intracellular Ca levels inhibited by TMB8. These results suggest that IL-13 causes phospholipase C activation and a release of Ca from InsP(3)-sensitive Ca stores. The regulation of phospholipase C is distinct for two of the known phospholipase families, the phospholipase C-beta and phospholipase C-. The phospholipase C-beta family is regulated by G proteins, while phospholipase C-1 and -2 are regulated by protein-tyrosine kinase (for review, see (43) ). The involvement of a protein-tyrosine kinase in the IL-13 signal transduction pathway was strongly suggested in a recent report. Indeed, genistein (one inhibitor of tyrosine kinase) completely blocked the IL-13-activated IL-4-dependent nuclear transcription factor in monocytic U937 cells(44) . A study concerning the suggested activation of phospholipase C- through tyrosine phosphorylation induced by IL-13 has already been undertaken in our laboratory.

Otherwise, we relate these initial events induced by IL-13 to the inhibition of the human monocyte biological response, the oxidative burst. Indeed, in the presence of TMB8 and neomycin sulfate, this inhibitory effect of IL-13 is impaired, whereas, in the absence of extracellular Ca or in the presence of indomethacin (inhibitor of cyclooxygenase pathway), IL-13 still induces a significant inhibition of ROI production. This last observation strongly suggests that prostaglandins, classically described to increase intracellular cAMP levels and inhibit ROI production in activated phagocytes(35, 37) , are not involved in IL-13 down-regulation of the biological response studied. Our results support the involvement of InsP(3)-dependent Ca mobilization and subsequent cAMP accumulation in the IL-13 inhibitory effect on ROI production.

In conclusion, we describe here for the first time the initial steps in the signaling pathway of IL-13 related to respiratory burst decrease. Indeed, direct measurement of second messengers and the use of metabolic inhibitors allowed us to establish a temporal sequence and a link between InsP(3)-dependent Ca mobilization, the cAMP protein-kinase pathway and inhibition of the protein kinase C-triggered respiratory burst in human monocytes pretreated with IL-13. The modulation of this important human monocyte function strengthens the notion that IL-13 could play a central role in the inflammatory process without totally inhibiting the monocyte oxygen-dependent anti-infectious capacity. Furthermore, work will be required to evaluate if the anti-inflammatory properties of IL-13 displayed in in vitro models, could be of clinical significance in the future for the treatment of inflammatory human diseases.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 33 61 32 29 67; Fax: 33 61 32 22 93.

(^1)
The abbreviations used are: IL, interleukin; ROI, reactive oxygen intermediates; TPA, 12-O-tetradecanoylphorbol-13-acetate; TMB8, 8[N,N-diethylamino]-octyl 3,4,5-trimethoxybenzoate, hydrochloride; Delta^9-THC, Delta^9-tetrahydrocannabinol; H89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide; CL, chemiluminescence; InsP(1), inositol 1-phosphate; InsP(2), inositol 1,4-bisphosphate; InsP(3), inositol 1,4,5-trisphosphate; InsP(4), inositol 1,3,4,5-tetrakisphosphate.

(^2)
Vita, N., Lefort, S., Laurent, P., Caput, D., and Ferrara, P.(1995) J. Biol. Chem., in press.


REFERENCES

  1. Minty, A., Chalon, P., Derocq, J. M., Dumont, X., Guillemot, J. C., Kaghad, M., Labit, C., Leplatois, P., Liauzun, P., Miloux, B., Minty, C., Casellas, P., Loison, G., Lupker, J., Shire, D., Ferrara, P., and Caput, D. (1993) Nature 362, 248-250 [CrossRef][Medline] [Order article via Infotrieve]
  2. McKenzie, A. N. J., Culpepper, J. A., de Malefyt, R., Briere, F., Punnonen, J., Aversa, G., Sato, A., Dang, W., Cocks, B. G., Menon, S., de Vries, J. E., Banchereau, J., and Zurawski, G. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 3735-3739 [Abstract]
  3. Zurawski, G., and de Vries, J. E. (1994) Immunol. Today 15, 19-26 [CrossRef][Medline] [Order article via Infotrieve]
  4. Cocks, B. G., de Waal Malefyt, R., Galizzi, J. P., de Vries, J. E., and Aversa, G. (1993) Int. Immunol. 5, 657-663 [Abstract]
  5. Punnonen, J., Aversa, G., Cocks, B. G., McKenzie, A. N. J., Menon, S., Zuraski, G., de Waal Malefyt, R., and de Vries, J. E. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 3730-3734 [Abstract]
  6. De Waal Malefyt, R., Figdor, C., Huijbens, R., Mohan-Peterson, S., Bennett, B., Culpepper, J., Dang, W., Zurawski, G., and de Vries, J. E. (1993) J. Immunol. 151, 1-12 [Abstract/Free Full Text]
  7. Montaner, L. J., Doyle, A. G., Collin, M., Herbein, G., Illei, P., James, W., Minty, A., Caput, D., Ferrara, P., and Gordon, S. (1993) J. Exp. Med. 178, 743-747 [Abstract]
  8. Denis, M., and Ghadirian, E. (1994) AIDS Res. Hum. Retroviruses 10, 795-802 [Medline] [Order article via Infotrieve]
  9. Hebert, J. M., Savi, P., Laplace, M. C., Lale, A., Dol, F., Dumas, A., Labit, C., and Minty, A. (1993) FEBS Lett. 328, 268-270 [CrossRef][Medline] [Order article via Infotrieve]
  10. Morel, F., Doussiere, J., and Vignais, P. V. (1991) Eur. J. Biochem. 201, 523-546 [Abstract]
  11. Segal, A. W., and Abo, A. (1993) Trends Biochem. Sci. 18, 43-47 [CrossRef][Medline] [Order article via Infotrieve]
  12. Bokoch, G. M., Quilliam, L. A., Bohl, B. P., Jesaitis, A. J., and Quinn, M. T. (1991) Science 254, 1794-1796 [Medline] [Order article via Infotrieve]
  13. Dusi, S., Della Bianca, V., Grzeskowiak, M., and Rossi, F. (1993) Biochem. J. 290, 173-178 [Medline] [Order article via Infotrieve]
  14. Savita, G., and Salimath, B. P. (1993) Cell. Signalling 5, 107-117 [Medline] [Order article via Infotrieve]
  15. Salimath, B. P., and Savitha, G. (1992) Cell. Signalling 4, 651-663 [Medline] [Order article via Infotrieve]
  16. Wu, Z., Wong, S. T., and Storm, D. R. (1991) J. Biol. Chem. 268, 23766-23768 [Abstract/Free Full Text]
  17. Verghese, M. W., Fox, K., Mc Phail, L. C., and Snyderman, R. (1985) J. Biol. Chem. 260, 6769-6775 [Abstract/Free Full Text]
  18. Ishitoya, J., and Takenawa, T. (1987) J. Immunol. 138, 1201-1207 [Abstract/Free Full Text]
  19. Pena, C., Poskus, E., and Paladini, A. C. (1980) Mol. Immunol. 17, 1487-1491 [Medline] [Order article via Infotrieve]
  20. Ziegler-Heitbrock, H. W. L., Ströbel, M., Kieper, D., Fingerle, G., Schlunck, T., Petersmann, I., Ellwart, J., Blumenstein, M., and Haas, J. G. (1992) Blood 79, 503-511 [Abstract]
  21. Forgue, M. F., Pipy, B., Beraud, M., Pinelli, E., Cambon, C., Didier, A., Souqual, M. C., and Vandaele, J. (1991) Carcinogenesis 12, 449-457 [Abstract]
  22. Olivier, M., Baimbridge, K. G., and Reiner, N. E. (1992) J. Immunol. 148, 1188-1196 [Abstract/Free Full Text]
  23. Kao, J. P. Y., Harootunian, A. T., and Tsien, R. Y. (1989) J. Biol. Chem. 264, 8179-8184 [Abstract/Free Full Text]
  24. Vita, N., Laurent, P., Lefort, S., Chalon, P., Lelias, J. M., Kaghad, M., Le Fur, G., Caput, D., and Ferrara, P. (1993) FEBS Lett. 335, 1-5 [CrossRef][Medline] [Order article via Infotrieve]
  25. Keppel, G. (1973) Design and Analysis: A Researcher's Handbook , Prentise Hall, Inc., Englewood Cliffs, NJ
  26. Gennaro, R., Florio, C., and Romeo, D. (1986) Biochem. Biophys. Res. Commun. 134, 305-312 [Medline] [Order article via Infotrieve]
  27. Myers, M. A., Mc Phail, L. C., and Snyderman, R. (1985) J. Immunol. 135, 3411-3416 [Abstract/Free Full Text]
  28. Wolfson, M., Mc Phail, L. C., Nasrallah, V. N., and Snyderman, R. (1985) J. Immunol. 135, 2057-2062 [Abstract/Free Full Text]
  29. Abramson, S. L., and Gallin, J. I. (1990) J. Immunol. 144, 625-630 [Abstract/Free Full Text]
  30. Lehn, M., Weiser, W. Y., Engelhorn, S., Gillis, S., and Remold, H. G. (1989) J. Immunol. 143, 3020-3024 [Abstract/Free Full Text]
  31. Zurawski, S. M., Vega, F., Jr., Huyghe, B., and Zurawski, G. (1993) EMBO J. 12, 2663-2670 [Abstract]
  32. Phillips, W. A., Croatto, M., and Hamilton, J. A. (1992) Biochem. Biophys. Res. Commun. 182, 727-732 [Medline] [Order article via Infotrieve]
  33. Doyle, A. G., Herbein, G., Montaner, L. J., Minty, A. J., Caput, D., Ferrara, P., and Gordon, S. (1994) Eur. J. Immunol. 24, 1441-14453 [Medline] [Order article via Infotrieve]
  34. Simchowitz, L., Fischbein, L. C., Spilberg, I., and Atkinson, J. P. (1980) J. Immunol. 124, 1482-1491 [Free Full Text]
  35. Takenawa, T., Ishitoya, J., and Nagai, Y. (1986) J. Biol. Chem. 261, 1092-1098 [Abstract/Free Full Text]
  36. Mitsuyama, T., Takeshige, K., and Minakami, S. (1993) Biochim. Biophys. Acta 1117, 167-173
  37. Kolb, J. P., Abadie, A., Paul-Eugene, N., Capron, M., Sarfati, M., Dugas, B., and Delespesse, G. (1993) J. Immunol. 150, 4798-4809 [Abstract/Free Full Text]
  38. Finney, M., Guy, G. R., Michell, R. H., Gordon, J., Dugas, B., Rigley, K. P., and Callard, R. E. (1990) Eur. J. Immunol. 20, 151-156 [Medline] [Order article via Infotrieve]
  39. Singh, V. K., and Leu, S. C. (1993) Immunol. Lett. 35, 239-246 [Medline] [Order article via Infotrieve]
  40. Baumgold, J., Paek, R., and Yasumoto, T. (1992) Life Sci. 50, 1755-1759 [CrossRef][Medline] [Order article via Infotrieve]
  41. Mac Neil, S., Lakey, T., and Tomlinson, S. (1985) Cell Calcium 6, 213-226 [Medline] [Order article via Infotrieve]
  42. Tang, W. J., and Gilman, A. G. (1992) Cell 70, 869-872 [Medline] [Order article via Infotrieve]
  43. Berridge, M. J. (1993) Nature 361, 315-325 [CrossRef][Medline] [Order article via Infotrieve]
  44. Köhler, I., Alliger, P., Minty, A., Caput, D., Ferrara, P., Höll-Neugebauer, B., Rank, G., and Rieber, E. P. (1994) FEBS Lett. 345, 187-192 [CrossRef][Medline] [Order article via Infotrieve]

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