After p~incubation with a 200 JIM concentration of the phos- phodiesterase inhibitor ..... R., Madariaga, J., and Simmons, R. L. (19921 J. Exp. Med. 176, 261-264.
THEJOURNALOF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 269, No. 13, Issue of April 1, pp. 9811-9816, 1994 Printed i n U.S.A.
Interleukin-4 Stimulates cGMP Production by IFN-y-activated Human Monocytes INVOLVEMENT OF THE NITRIC OXIDE SYNTHASE PATHWAY* (Received for publication, August 2, 1993, and in revised form, December 2, 1993)
Jean Pierre KolbSO, Nathalie Paul-Eugenem, Chantal Damaisll, Kunio YamaokaS, Jean Claude DrapierS, and Bernard Dugasn From the Slnstitut National de la Sante et de la Recherche Medicale U365, Znstitut Curie, lpnstitut National de la Sante et de la Recherche Mbdicale U313, CHU Pitie-Salpgtriere, Paris, and nlnstitut National de la Sante et de la Recherche Medicale ICJF 92-10, Montpellier, France
uce reactive oxygen species (2-6); these effects are correlated Resting human blood monocytes from some donors were found to produce a small amountof 3‘-5’ guanine with a decrease in their tumoricidal activity (2). The second messengersassociated to IL-4 signalling are cyclic monophosphate (cGMP) in response to interleupoorly understood and appear t o be different between human A much higher response was observed when kin 4 (IL-4). monocytes were preincubated with interferon (IFN-y), and mouse. The IL-4 receptor has been cloned and belongs to which alone was ineffective. Preincubation of mono- the superfamily of hematopoietin receptors, which neither discytes with IL-4 led, in contrast, to their subsequent in- play the canonical structure of receptors linkedto GTP-binding capacity to generate cGMP in response to IL-4. The ac- proteins nor exhibitintrinsic kinaseactivity in theirintracytoplasmic domain (7). Unlike other hematopoietins, IL-4 does not cumulation cGMP of induced by IL-4 in IFN-y induce ~ 2 1 “activation, ~ an event involved in the downstream preincubated monocytes was dose-dependent and peaked about 15 min afterits addition. It was inhibited cascade of tyrosine kinase-associated receptors (8). Moreover, W-mono-methyl-L-arginine (L- at variance with the murine system, IL-4-induced changes in in the presence of NMMA), an inhibitor of the nitric oxide synthase path- the phosphorylation pattern of human cells have been difficult way. This suppressive effect of L - N M M A was revertedby to detect (9). Finney et al. (10) reported that IL-4 stimulated an an excess of L- but not of D-arginine. Accumulation of early phosphoinositide hydrolysis and calcium mobilization in cGMP was significantly reduced by addition of soluble resting human B cells, followed by a delayed increase in CAMP, guanylyl cyclase inhibitors, such as LY83853 and meth- both events being necessary for the induction of CD23. Reylene blue, butwasnotimpairedin the presence of cently, we observed that IL-4 also induced a delayed accumuEGTA, suggesting that the pathway involvedis calcium lation of CAMPin monocytes, which peaked about 20 min after independent. In addition,IL-4 induced an increasedse- its addition. This late CAMP increase suggested therefore an cretion of nitrite by monocytes, that was potentiated by indirect mechanism of action of IL-4. This prompted us to test IFN-./ and inhibited by L - N M M A . Taken together, these a possible action of IL-4 on the cGMP system, through the results suggest that the sequential exposure of mono- generation of nitric oxide. cytes to IFN-./ and IL-4 elicits the release of NO from Nitric oxide (NO) is a short-lived radical that hasbeen idenL-arginine, which in turn is capable to stimulate soluble tified in recent years as a pleiotropic mediator. NO is syntheguanylyl cyclase. sized from L-arginine by nitric oxide synthases (NOS), which are expressed in different cell types eitheras constitutive (cNOS) or inducible (iNOS)enzymes. Cytokines, suchas IFN-y, Interleukin-4 (IL-4)’ is a 20-kDa glycoprotein which displays and microbial components, suchas lipopolysaccharide, activate a broad spectrum of biological activity. It ismostly produced by the nonspecific resistance of murine macrophages against activated T lymphocytes (Th,), but also by mast cells, basophils, pathogens ( l l ) , in part by stimulating the expression of an and other cell types (1).IL-4 acts on several cells of the hema- iNOS in these cells (12). At variance with the results obtained topoietic lineage. It induces the differentiation of human mono- with rodents macrophages, IFN-y is unable, however, to trigger cytes into macrophage-like cells type in uitro, down-regulates NOS activation in human monocytes/macrophages. The existtheir release of several pro-inflammatory mediators and, in ence of an inducible NOS in man, once a controversial issue, contrast to the murine system,reduces their capacity to prod- has now been demonstrated in several cell types (13-16). It is clear now that this pathway is at the origin of the hypotension * This work was supported by the Institut National de la Sante et de of patients with a septic shock (17) andof the augmentation of la Recherche Medicale andby grants from Ministere de la Rechercheet nitrite in patients undergoing IL-2 therapy (18, 19). In addiTechnologie and Association pour la Recherche sur le Cancer. The costs tion, Salvemini et al. (20) reported that platelet aggregation of publication of this article were defrayed in part by the payment of page charges. Thisarticle must therefore be hereby marked “advertise- was inhibited by a NO-like factor released by human neutroment” in accordance with 18U.S.C. Section 1734 solely to indicatethis phils and mononuclear cells. Yet, several works suggested that fact. the microbicidal activity of cytotoxic macrophages was inde0 To whom correspondence shouldbe addressed: INSERM U365,Inst. pendent of NO production (21-23), and there is stillno definiCurie, 26 rue dUlm, 75231, Paris Cedex 05, France. Tel.: 1-40516719; tive evidence that the NO pathwayis operative in human Fax: 1-44070785. The abbreviations used are: IL-4, interleukin 4; FCS, fetal calf se- monocytic cells. rum; cGMP, 3’-5’ guaninecyclicmonophosphate;PBMC,peripheral In the present work, we report that IL-4 triggers an early blood mononuclear cells; NO, nitric oxide; NOS, nitric oxide synthase; SNP, sodiumnitroprusside; IFN, interferon; IBMX, Isobutylmethylxan- cGMP increase in monocytes that is markedly potentiated by thine; L-NMMA,p-mono-methyl-L-arginine; TNF, tumor necrosis fac- prior stimulation of these cells by IFN-y. This mechanism intor; MEM, minimal essential medium. volves the L-arginine-dependent pathway of NO production, as
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IL-4 Diggers cGMP in IFN-y-actiuated Monocytes via L-Arginine Pathway
evidenced by its inhibition with L-NMMA. In addition, a late accumulation of nitrite, inhibited by L - N M ~was , detected in supernatants of monocytes stimulated by IFN-y and IL-4. Our data therefore suggest that NOS activation can be achieved in human monocytes through this combination of cytokines. EXPERIMENTAL PROCEDURES
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Isolation of Human Monocytes-Human peripheral blood mononuclear cells (PBMC) were isolated from normal donors as described (24). Briefly, blood samples were centrifuged (900 rpm, 10 min) to reControl IFN-gamma 1L-4 move platelets. Cells were diluted in RPMI 1640 medium (Bioproduct, PREINCUBATION Les Ulis, France), and PBMC were harvested after centrifugation (2000 rpm, 20 min) on a Ficoll-Hypaque (Pharmacia, Uppsala, Sweden)graFIG.1. IL-4 stimulates cGMF accumulation in IFN-y-activated dient. After incubation of PBMC (1x IO7 celldml) in Petri dishes (Nunc,monocytes. Monocytes in RPMI 1640 medium + 5% FCS (2 x lo6 Rocksille, Denmark) for 1 h at 37 "C i n RPMI 1640 medium with 10% celldml) were cultured for 48 h with medium alone or with IL-4 (10 nglml) or IFN-y (lo3 unitsiml), then recovered and resuspended in FCS (Gibco, Paisley, United Kingdom), m o n ~ were ~ s harvested by scraping plates with a rubber policeman in cold phosphate-buffered RPMI 1640 medium(7 x loficellslml). For each group, cells were divided saline solution containing1nm EDTA. Cells were more than 95% viable in two parts which were incubated(+) or not (-1 for 3 h with L-NMMA (1m).IBMX (200 PI) was added during the last 30 of min incubation monocytes as assessed by trypan blue exclusionand nonspecific ester- before stimulation with control mediumor IL-4 (50 ng/ml) for 15 min. ase staining. Lymphocytes and granulocytes never exceeded 5% of the Cell extracts were analyzedfor cGMP accumulation by radioimmunototal. assay. As a positive control, sodium nitroprussideSNP (500 JIM) induced Monocyte Culture-Monocytes were cultured (2 x lo6 cells/ml) in 5.1 pmol of cGMP/106 cells. Data are mean -c S.E. from triplicate plastic Petri dishes (Nunc), in RPMI 1640 medium with 2 mM fresh samples inone experiment representativeof five. The significanceof the glutamine, 1 mM sodium pyruvate,penicillin (100 unitdml), streptomy- results was analyzedaccording t o the Student's t test for small samples cin (100pg/ml), and5% FCS (Gibco, Paisley, United Kingdom). Medium (*). * , p < 0.05. **, p < 0.001, was endotoxin-free by the limulus amoebocyte lysate assay (E-toxate, Sigma). Cells were incubated (48 h, 37 "C) under a 5% CO,, 95%air, and RESULTS 100% humidity atmosphere, withor without various reagents, washed, P o t e n ~ ~ a t ~ of o IL-4-induced n c ~ M P A c c u ~ u Z a t i oinnHuman and suspended a t t h edesired concentration in RPMI 1640 medium to bys IFN-y Preincubation-In a first set of experiperform tests of stimulation for cyclic nucleotide determination. Plate- ~ o n o c ~ t e ments, IL-4 was found to triggera slight butsignificant cGMP lets were undetectableat the end of culture. ~eagents-Isobutylmethylxanthine (IBMX), manganese superoxide increase in freshly isolated monocytes for three out of seven dismutase, methylene blue,EGTA, NaNO,, catalase, and other chemi- donors tested, whereas no significant augmentation over the cals were from Sigma. LY 83853 (6-anilino-5,8-quinolinedione) was a basal level was observed for the four other donors. In the rekind gift of C. Pellat (Institut National de la Sante et de la Recherche sponding monocytes, IL-4-induced cGMP accumulation (about Medicale, U365, Institut Curie). Recombinant human IL-4 from Es2-fold increase) peaked 10-15 min after addition of IL-4 (200 cherichia coli (specific activity: 1 x io7 unitdmg; 1 unit = 10 ng) was purchased from Immugenex (Los Angeles, CAI. Recombinant bacterial- units/ml) andwas totally abolishedby a prior 3-h incubation of derived IFN-.I (specific activity: 4 x lo6 IU/mg) was a generous gift of monocytes with 1 mM L-NMMA, a competitive inhibitor of LRoussel-Uclaf (Romainville, France).L-NMMAand p-nitro-L-arginine, arginine for the NOS pathway (not shown). two N-guanido-substituted analogues of L-arginine that arecompetitive Inasmuch as only monocytes from some donors displayed a inhibitors of NOsynthase,werepurchased from Calbiochem and significant cGMP responsewhenchallengedwith IL-4, we Sigma, respectively, and were used at 0.5-1 mM final concentration. speculated that it may result from difference in theactivation Determination ofcGMP-For measuring cytoplasmiccGMP levels in whole cells, monocytes were resuspended (7 x 1O6/ml)in RPMI 1640 state of these cells in uiuo. Since IFN-y is one of the major medium. After p~incubation with a 200 JIM concentration of the phos- cytokines which affects m o n ~ y t ephenotype and function, we phodiesterase inhibitorIBMX (37 "C, 15 m i d , 1 x loGcells (135$) were investigated the effect of preincubating monocytes with IFN-y distributed inEppendorf microtubes,in duplicate or triplicate samples. before IL-4 stimulation. Monocytes were incubated for 48 h Fifteen pl of the different ligands were added for various periods of with or without IFN-y (lo3 unitdml), then stimulated with incubation a t 37 "C in a water bath. Reactions were stopped by addition IL-4. As shown on the representative experiment depicted in of 25 pl of perchloric acid (35%) and immediatecooling. After 15 min at Fig. 1, a much higher cGMP accumulation was observed in 0 "C, tubes were spun (3000 x g, 15 rnin), pellets were discarded, and supernatants neutralized withKOH. KCIO, precipitates were removed IFN-y-treated monocytes in comparison with monocytes culby centrifugation at 4 "C (3000 x g, 15 rnin). Neutralized supernatants tured in medium alone. With a single exception, this potentiawere kepta t -70 "C until cGMP determination. After acetylationof the tion of the IL-4 response by IFN-y was observed for all donors, samples, cGMP levels were determined by specific radioimmunoassay, including those who did not respond to IL-4 alone. Again, inaccording to the specificationsof the manufacturers (kits from Amer- creases incGMP levels were inhibited with L-NMMA.Noteworsham France,Les Ulis or from NEN, DuPont, Dreieich, Federal Repub- thy, monocytes incubated for 48 h with IL-4 (10 ng/ml) did not lic of Germany). In some instances, the amount of cGMP released in respond any more to a restimulation with IL-4 (Fig. 1).Yet, culture medium was also estimated from cell-free supernatants collected after various timesof incubation. The significance of the results IL-4-treated monocytes expressed the CD23 (FccR,) antigen was analyzed by the Students's t test adapted for small numbers of and could be triggered to increased cGMP accumulation through ligation of CD23 with specific mAb or IgE complexes.' samples (25). Of interest, IFN-yalone did not induceany significant cGMP Determination of NO+"ure supernatants were assayed for the stable end productof NO, NO, according to previously described Griess increase in monocytes preincubated for 48 h with either mereaction adapted from Green et al. (26).Briefly, cell-free supernatants dium alone, IFN-y,or IL-4 (not shown). The enhancingeffect of (50 pl), in triplicate samples, were mixed in wells the of a 96-well mul- IFN-y on IL-4-induced cGMP generation was already detectatititration plate with 100 ofplthe Griess reagent, made of a U1 mixture ble after 1 day of incubation of the monocytes with IFN-y, of 1%(w/v) sulfanilamide in30% acetic acid and 0.5% (w/v) ofN-1-naphtylethylenediamine dihydrochloride in60% acetic acid. Thiscompound peaked a t day 2, then sharplydeclined (not shown). IL-4 induces a Dose- and Time-dependent eGMP Accumulareacts with nitrite t o form a chromophore that was detected spectroa Titertek multiskan (Flow) reader. The photometrically (540 nm) using concentration of nitrite was calculated using calibration withconknown N. Paul-Eugene, J. P. Kolb, M. Arock, F. Ouaaz, M. Sarfati, P. Gracentrations of NaNO,. Significance of the results was analyzedby the ber, J. Y. Bonnefoy, P. Debre, D. M. Mossalayi, andB. Dugas, submitted for publication. modified Students's t test for small number of samples (25).
cr?
IL-4 niggers cGMP in IFN- y-activated Monocytes via L-Arginine Pathway
0
20
40
60
80
9813
100
IL-4 (nglml)
FIG.2. IL-4 induces a dose-dependent cGMP accumulation in IFN-y-stimulated monocytes. Monocytes (2.5 x 106/ml) were incubated for 48 h in RPMI 1640 medium+ 5% FCS with IFN-y (lo3units/ ml), then resuspended in RPMI 1640 medium (2 x lo6 celislml), incubated for 30 min a t 37 "C with IBMX (200 PM),and stimulated for 15 min in the presence of various concentrations of IL-4. Accumulation of cGMP was determined as previously, using the EL4 test o f h e r s h a m . Data are mean ? S.E. from duplicate samples in one experiment representative of three. SNP, sodium nitroprusside, 500 PM.
tion in IFN-y-preactivated Monocytes-The IL-4-induced cGMP accumulation in IFN-y-stimulatedmonocytes is concentrationdependent, as shown on Fig. 2. A slight increase was already observed at 1ng/ml and maximum stimulation was reached for 50-100 ng/ml IL-4. A plateau of stimulation was observed for concentrations above 100 ng/ml (not shown). The kinetics of induction by IL-4 of cGMP in IFN-y-stimulated monocytes was also analyzed in comparison with sodium nitroprusside (SNP), a chemical NO donator. SNP elicited a rapid and sustained augmentation of cGMP, whereas theaccumulation triggered by IL-4 peaked about 15 min afteraddition of the interletkin, then started todecline slowly (Fig. 31. Involvement of the NO Synthase Pathway in IL-4-induced cGMP Accumulation-The reversal of IL-4-induced cGMP increase inmonocytes in thepresence of W-methyl-mono-L-arginine is a strong argument for the implication of the NO synthase pathway in this process. This conclusion was further reinforced by the observation that the inhibitory effect of LNMMA could be alleviated in thepresence of a 5-fold excess of L-arginine, but not D-arginine, as reported in Table I. In contrast, neither L-NMMAnor L-or D-arginine interfered with the cGMP accumulation generated in thepresence of SNP. Soluble guanylyl cyclase are heterodimers containingheminic prosthetic groups thatare stimulated by NO releasing compounds. Addition of LY83583, a n inhibitor of soluble guanylyl a marked impairment cyclase activation, wasfound to result in of IL-4-triggered cGMP accumulation, as shown on Fig. 4 (top). A 65% inhibition was obtained at lo-' M LY83583 and a near M. However, background level wasnot totalsuppression at affected by this inhibitor, suggesting that anothermechanism is responsible for the homeostasis of basal cGMP level. Similar results were obtained in thepresence of 10 PM methylene blue, another inhibitor of soluble guanylyl cyclase activation (not shown). The potentiating effect of IFN-y preincubation suggested thatIL-4 might be acting on a n inducible NO synthase fiNOS). Murine macrophage-inducible NO synthase, as well as other iNOS, are not regulated by intracytoplasmic calcium increase, as opposed to the cNOS present in brain andendothelial tissue.Inagreementwiththis hypothesis, theincrease of cGMP elicited by IL-4 was not modified when monocytes were preincubated with EGTA, a chelator of calcium (Fig. 4, bottom). This augmentation of cGMP was also not affected in the presence of two inhibitors of calciudcalmodulin complex, W-7, and calmidazonium (not shown). NO half-life is markedly shortened in the presence of superoxide radical 0: When Mn superoxide dismutase, which catalyzes the conversion of 0, into 0, and H,O,, was added to the
0' 0
20
10
30
TIME (rnin) FIG.3. Kinetics of IL-4-induced cGMP accumulation in IFN-ystimulated monocytes. Monocytes, incubated for 48 h with IFN-y (IO3 unitdml) as previously, weretreated or not for 3 h with t-NMMA(1 mM), then challenged for different times of stimulation with IL-4 (50 ngiml) or SNP (500 p ~ fData . are mean c S.E. from duplicate samples in one experiment representative of three. TABLE I IL-4triggers cGMP production in IFN-y Stimulated monocytes
through activation o f t h e NO pathway Monocytes wereincubated for 48 h with IFN-y (IO3unitdml), washed, and resuspended at 7 x lo6 celldml in RPMI 1640 medium with or without 1mM L-NMMA,together with or without 5 mM D- or L-arginine, for a 2-h incubation at 37 "C. The cells werethen stimulated for 15 min with medium, IL-4 (100 units/ml), or sodium nitroprusside (500 p). Perchloric extracts were then analyzed for cGMP content by RIA with acetylation of the samples, as described under "Experimental Procedures." Data are mean S.E. from triplicate samples in one experiment representative of three. NS, not significant. L
Stimulus
-
( 1mM)
N
~Arginine ~ 15 mM)
cGMP
P
" .
-
Medium
+
+ + IL-4 (10 ng/ml)
-
+ + +
SNP (500 p
~ )
-
+
+ + -
-
L
D
L 0
L
D L.
D
fmot i 106' cells 80 c 10
97c 12 88 11 14 104? 280 e 17 105 c 15 265 c 19 97 e 6 1805 t 132 1670 c 117 1840 t 75 1585 c 105 1720 95 1657 c 110
2
S.E.
NS NS NS NS
NS
