DIFFERENTIAL REGULATION OF INTERLEUKIN4 AND INTERLEUKIN 13 PRODUCTION BY

INTERFERON a

Arthur Kaser, Clemens Molnar, Herbert Tilg

Interferon a (IFN-a) has proven its clinical usefulness in a variety of diseases of diversepathogenesis. In addition to direct antiviral effects, recent evidence suggests that its interactionwith the cytokine cascade might contribute to its mechanism of action. This study wasundertaken to determine whether IFN-a influences the synthesis of interleukin 4 (IL-4) andIL-13, two cytokines which share many biological properties on various cells and tissues andwhich have a profound role in regulating immunological and inflammatory responses. Peripheralblood mononuclear cells (PBMC) from healthy volunteers were stimulated with ConcanavalinA (ConA), phorbol myristate acetate (PMA), anti-CD3/CD28 mAbs, either alone or in variouscombinations, and incubated with increasing concentrations of IFN-a. IL-4 and IL-13 mRNAwas determined by Northern hybridizations and IL-4 and IL-13 protein synthesis was evaluatedby specific enzyme-linked immunosorbent assay (ELISA). IFN-a led to a profound decrease ofIL-13 mRNA expression after an incubation period of 5 h with ConA alone or in combinationwith PMA, whereas it showed no regulatory effect on IL-4 mRNA expression. After anincubation period of 24 h, the decrease in IL-13 mRNA expression after addition of IFN-a waseven more pronounced. At the protein level, IFN-a increased IL-4 synthesis dose dependentlyregardless of the mode of activation. This increase was most pronounced after stimulation withConA or anti-CD28/PMA. In contrast, IL-13 synthesis was strongly downregulated by IFN-ain a dose-dependent manner irrespective of the activating agent. It is concluded that IL-4 andIL-13, although showing similar biological effects, are differentially regulated by IFN-a.

7 1998 Academic Press Limited

Interferon a (IFN-a) is clinically useful in a varietyof diseases of diverse pathogenesis including chronicviral hepatitis, hairy cell leukaemia and papillomas.1,2

Despite its clinical success, the detailed mechanisms ofaction have not yet been well defined. Besides its directantiviral effects, IFN-a interacts with the cytokinecascade. Although primarily regarded as a pro-inflam-matory cytokine, IFN-a has been recently shown toexhibit several immunomodulatory and anti-inflamma-tory activities in vitro and in vivo. These include theinhibition of the synthesis of interleukin 1 (IL-1),3,4

IL-5,5 IL-8,6,7 and granulocyte-macrophage colony-stimulating factor (GM-CSF),4 whereas the productionof IL-1 receptor antagonist (IL-1Ra),8 IL-109 and thesoluble form of the tumour necrosis factor p55 receptor(sTNF-R55) is augmented.10

IL-4 is a polypeptide cytokine that is mainlysecreted by Th2 cells.11 It enhances the proliferative

response of B cells and induces class switching toIgG4 and IgE. IL-4 exhibits growth-promotingactivities on T cells, plays an important role inestablishing Th2 responses and acts as an autocrinegrowth factor for Th2 T cell clones.12 On monocytes,IL-4 reduces their antibody-dependent cellular cyto-toxicity (ADCC), whereas it enhances their ability topresent antigen.13 IL-4 also inhibits the synthesis ofpro-inflammatory cytokines and increases the synthesisof IL-1Ra.14

IL-13 is a cytokine which is produced by CD8+

and CD4+ T cell clones of Th0, Th1 and Th2 type inresponse to antigen-specific or polyclonal stimuli.15 TheIL-13 gene is located on chromosome 5 band q31 inclose proximity to the IL-4 gene, and in the samecluster of genes IL-5, IL-3 and GM-CSF are encoded.16

IL-13 resembles IL-4 in its activities on B cells andmonocytes, whereas it has no growth-promotingeffect on T cells and cannot compete for IL-4binding to a human T cell line.17 Recently an inhibitoryeffect of IL-13 on IL-8 and RANTES-inducedchemotaxis of CD4+ and CD8+ T cells has beendescribed, indicating that T cells do respond to IL-13.18

Production of many LPS-induced cytokines, such asIL-1a, IL-1b, IL-6, IL-8, IL-10, IL-12, TNF-a,macrophage inflammatory protein-1a (MIP-1a), GM-CSF, and granulocyte CSF, is inhibited by IL-13,

From the Department of Medicine, University Hospital Innsbruck,Innsbruck, Austria

Correspondence to: Herbert Tilg, Department of Medicine,University Hospital Innsbruck, Anichstr. 35, 6020 Innsbruck,Austria

Received 26 March 1997; accepted for publication 11 July 19977 1998 Academic Press Limited1043–4666/98/020075+07 $25.00/0/ck970270

KEY WORDS: interferon a/interleukin 4/interleukin 13

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whereas IL-1Ra is upregulated.19,20 These properties areshared with IL-4. Therefore, both IL-4 and IL-13 haveanti-inflammatory activities.

The present experiments were designed toinvestigate the role of IFN-a on the regulation of IL-4and IL-13 production in peripheral blood mononuclearcells (PBMC).

RESULTS

Regulation of IL-4 mRNA expression by IFN-aThe effect of IFN-a on the expression of IL-4

mRNA was studied in human PBMC. IFN-a did notinduce detectable levels of IL-4 mRNA after 5 h ofincubation (data not shown). Subsequently, PBMCwere stimulated with ConA alone for 5 h and incubatedwith concentrations of IFN-a between 0 and10 000 IU/ml. A 5-h stimulation with ConA causedexpression of IL-4 mRNA, but IFN-a did have noregulatory effect (data not shown). Stimulation ofPBMC with ConA and PMA instead of ConA aloneled to a stronger expression of IL-4 mRNA comparedto ConA alone, but IFN-a again showed nomodulatory effect (Fig. 1). After stimulation withConA and PMA for 24 h, we were unable to detect anyIL-4 mRNA (Fig. 2).

Regulation of IL-13 mRNA expression by IFN-aIncubation of PBMC with various concentrations

of IFN-a for 5 h did not induce IL-13 mRNA

Figure 2. Differential effects of IFN-a on IL-4 and IL-13 mRNAexpression induced by ConA (10 mg/ml) and PMA (1 ng/ml) in PBMC.

PBMC were incubated in medium alone (lane 1), or stimulated withConA and PMA (lanes 2–7) in the absence (lane 2) or presence of1, 10, 100, 1000 and 10 000 IU/ml IFN-a (lanes 3, 4, 5, 6, 7,respectively). After 24 h, total RNA was extracted and hybridizedwith IL-4, IL-13 and GAPDH probes.

expression (data not shown). After stimulation withConA and incubation for 5 h, a weak IL-13 mRNAmessage was detectable, and increasing concentrationsof IFN-a led to a reduction of IL-13 mRNA (data notshown). Stimulation with ConA and PMA andincubation for 5 h led to a more pronounced IL-13mRNA message. Increasing concentrations of IFN-aresulted in a strong reduction of IL-13 mRNA levels(Fig. 1). In experiments with an incubation period of24 h and stimulation with ConA and PMA, weobserved the strongest reduction of IL-13 mRNAmessage by increasing concentrations of IFN-a withthe maximum effect at the highest concentration tested(Fig. 2).

Regulation of IL-4 synthesis by IFN-aTotal production of IL-4 (secreted and cell-associ-

ated) was assessed in PBMC cultures after 48 h ofincubation. IFN-a alone did not induce IL-4 synthesis(data not shown). In ConA stimulated PBMC, IFN-aincreased the production of IL-4 (Fig. 3A), whereasafter stimulation with ConA and PMA IFN-a did onlyslightly affect IL-4 levels (Fig. 4A).

In subsequent experiments, we induced cytokinesynthesis by monoclonal antibodies against CD3 andCD28, or with a combination of anti-CD28 with PMA.As shown in Fig. 5A, we could detect an increase ofIL-4 synthesis in anti-CD3/CD28 stimulated PBMC byincreasing concentrations of IFN-a.

In PBMC stimulated with a combination ofanti-CD28 mAbs and PMA and incubation over 48 h,

Figure 1. Differential effects of IFN-a on IL-4 and IL-13 mRNAexpression induced by ConA (10 mg/ml) and PMA (1 ng/ml) in PBMC.

PBMC were incubated in medium alone (lane 1), or stimulated withConA and PMA (lanes 2–7) in the absence (lane 2) or presence of1, 10, 100, 1000 and 10 000 IU/ml IFN-a (lanes 3, 4, 5, 6, 7,respectively). After 5 h, total RNA was extracted and hybridized withIL-4, IL-13 and GAPDH probes.

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we were also able to show a significant increase of IL-4synthesis by increasing concentrations of IFN-a, witha maximum effect at 10 000 IU/ml (Fig. 6A). This effectwas statistically significant even at a concentration of1 IU/ml IFN-a.

Regulation of IL-13 synthesis by IFN-aIn unstimulated PBMC, IFN-a did not induce

IL-13 protein synthesis after an incubation period of48 h (data not shown). Stimulation with ConA orConA plus PMA induced IL-13 synthesis, which wasmarkedly reduced by increasing concentrations ofIFN-a (Fig. 3B and 4B). In the experiments depictedin Fig. 5B, we could show that IFN-a leads to adose-dependent reduction of IL-13 synthesis in PBMCafter stimulation with anti-CD3/CD28 mAbs. Themaximum effect was observed at the highest concen-tration tested. Stimulation of PBMC with a combi-nation of anti-CD28 mAbs and PMA and incubationwith increasing concentrations of IFN-a leads also toa comparable reduction of IL-13 synthesis (Fig. 6B).

DISCUSSION

In the present study, we describe the differentialregulation of synthesis of IL-4 and IL-13 in PBMC byIFN-a. IL-4 and IL-13 share many biologicalproperties. They have been shown to affect B cell andmonocyte function. Both cytokines induce the pro-duction of IgM, IgG and IgE by B cells when theappropriate second signal is provided by activated Tcells, membranes prepared from activated T cells orCD40 ligand expressing cells.21,22 IL-4 and IL-13induce identical changes in morphology and adherenceof human monocytes, downregulate the expression ofCD14, Fcg receptors and enhance the expressionof various adhesion molecules, as well as class II MHCantigens.23 Both IL-4 and IL-13 induce the expressionof CD23 on B cells and monocytes.22 Furthermore,both IL-4 and IL-13 have anti-inflammatory activitiesthrough their inhibitory effects on the production ofpro-inflammatory cytokines IL-1, IL-6, IL-8, MIP-1a,TNF-a, as well as IL-12 p35 and IL-12 p40 andthrough their enhancing effects on the production ofIL-1Ra.19,20,24,25 While IL-4 receptors are expressedon virtually every cell type studied so far, the receptorpattern of IL-13 is possibly more restricted, as a T cellline does not express receptors for IL-13 and does notrespond to IL-13 with a proliferative response,15

although recently it has been shown that IL-13 altersthe chemotactic response of CD4+ and CD8+ T cellsinduced by IL-8 and RANTES.18 IL-4 in contrast toIL-13 promotes the growth of activated T cells and Tcell clones26 and also induces the expression of CD8 onCD4+ T cell clones.23,27

Figure 3. Differential effects of IFN-a on the production of IL-4 (A)and IL-13 (B) in PBMC from healthy individuals.

PBMC (5×105 cells per well) obtained from different donors werestimulated with ConA (10 mg/ml) in the absence or presence ofgraded concentrations of IFN-a. Controls left unstimulated did notshow any IL-4 or IL-13 synthesis. After 48 h, cells were frozen andthawed three times and the lysates were assayed for IL-4 and IL-13by ELISA. Mean 2 SEM of nine experiments are shown. *PQ 0.05compared with 0 IU/ml IFN-a.

There is evidence that IL-4 and IL-13 play adifferential role in B cell development at different stepsof B cell ontogeny.28 IL-13 failed to induce germ-line omRNA synthesis in sm− pre-B cells, while IL-4 could

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Figure 4. Differential effects of IFN-a on the production of IL-4 (A)and IL-13 (B) in PBMC from healthy individuals.

PBMC (5×105 cells per well) obtained from different donors werestimulated with ConA (10 mg/ml) and PMA (1 ng/ml) in the absenceor presence of graded concentrations of IFN-a. Controls leftunstimulated did not show any IL-4 or IL-13 synthesis. After 48 h,cells were frozen and thawed three times and the lysates were assayedfor IL-4 and IL-13 by ELISA. Mean 2 SEM of six experiments areshown. *PQ 0.05 compared with 0 IU/ml IFN-a.

that TCR-stimulated naive human CD4+45RO− T cellsdevelop into effector cells that secrete IL-13, IL-5 andIFN-g, but not IL-4, and help efficient IgE productionby B cells.29 These data together suggest that IL-4 andIL-13 affect B cell function in a different manner.

Although IL-4 resembles IL-13 in many biologicalproperties, the regulation of these cytokines seems to bedifferentially regulated. It has been previously shownthat IL-13 expression can be induced by ConA, PMA,phytohaemagglutinin (PHA), the Ca2+ ionophoreA23187 or anti-CD3 alone, while in contrast, inductionof IL-4 needed costimulation with the exception ofA23187.15 Additional to these differential effects ofvarious activation-inducing agents on IL-4 and IL-13synthesis, we now report that IFN-a differentiallyregulates the expression of IL-4 and IL-13 in stimulatedPBMC. In accordance with previous reports,15 we showthat IL-4 mRNA expression is only transientlydetectable compared to IL-13 mRNA expression, asIL-4 mRNA was no longer detectable after 24 h ofincubation, whereas IL-13 mRNA was still stronglyexpressed. This could possibly mean that IL-4expression is more tightly regulated than IL-13expression. Interestingly, it has been recently shownthat CD3 engagement of CD4+ or CD8+ T cellsnegatively influences IL-13 production, which can bereversed by cyclosporin A (CsA), whereas IL-4production in CD4+ T cells requires CD3 costimula-tion and addition of CsA inhibits IL-4 synthesis.30 Inaccordance with this observation, we show thatstimulation with anti-CD28 mAbs and PMA leads to amore pronounced IL-13 synthesis than stimulation viaanti-CD3/anti-CD28 mAbs. These data on thedifferences in kinetics and the observation that IL-4and IL-13 are not co-ordinately regulated by T cellactivators or by IFN-a, indicate that IL-4 and IL-13genes are independently regulated, despite their closelinkage on chromosome 5, which increases thepossibility that these two cytokines may share commonregulatory regions.31

It has been previously shown that IFN-a increasesthe frequency of IFN-g-producing CD4+ T cells whilethe frequency of IL-4 producing cells remainsunchanged, suggesting that IFN-a favours the ex-pression of a Th1 phenotype.32 Our observation thatIFN-a augments the production of IL-4 is remarkable,since IL-4 has been shown to suppress the productionof IFN-g by PBMC.33 This enhancing effect of IFN-aon IL-4 is in accordance with a recent report statingthat IFN-a augments the secretion of IL-4 by CD4+ Tcells.5

IFN-a is primarily considered to be a pro-inflam-matory cytokine, but recently it has been shown thatIFN-a possesses several anti-inflammatory properties,which could be regarded as a self-limiting mechanismof inflammation induced by IFN-a.34 Since we now

induce o mRNA synthesis in these pre-B cells,suggesting that IL-13 does not regulate pre-B celldifferentiation.28 On the other hand, there is evidence

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show that IFN-a enhances the production of IL-4,which has various anti-inflammatory properties, thiscould be another way to limit inflammatory responses.On the other hand, we show that IL-13 productiondiminishes with increasing concentrations of IFN-a,suggesting that potential anti-inflammatory propertiesof IFN-a are not mediated via this cytokine.

MATERIALS AND METHODS

ReagentsRecombinant human (rh) IFN-a2b was obtained from

Schering-Plough (AESCA, Traiskirchen, Austria). An anti-CD3 monoclonal antibody (CLB-T3/4.E) of IgE-isotype aswell as an anti-CD28 monoclonal antibody (CLB-CD28/1)was purchased from CLB (Diagnostica, Vienna, Austria).Concanavalin A (Con A) and phorbol 12-myristate13-acetate (PMA) as well as Polymyxin B sulfate were fromSigma Chemical Co. (Vienna, Austria). FCS was fromGIBCO (Life Technologies, Schoeller Pharma, Vienna,Austria).

Isolation of PBMCPeripheral blood mononuclear cells (PBMC) were

isolated from the heparinized blood of healthy volunteers bydensity gradient centrifugation through Ficoll-Hypaque(Sigma). The cells were washed three times in RPMI 1640(GIBCO Life Technologies) and the indicated number ofcells was used for stimulation of cytokine synthesis forsubsequent IL-4 and IL-13 measurement by enzyme-linkedimmunosorbent assay (ELISA) and corresponding Northernhybridizations.

Stimulation of cytokine synthesisIn experiments with stimulation of cytokine synthesis by

ConA or ConA in combination with PMA, isolated PBMCwere cultured in polypropylene tubes at a density of 5×105

cells/ml in a final volume of 1 ml RPMI 1640 supplementedwith 5% FCS and 5 mg/ml polymyxin B sulfate (PMB). Foranalysis of cytokine synthesis induced by anti-CD3/anti-CD28 and anti-CD28/PMA, 2×105 cells were cultured in afinal volume of 0.5 ml RPMI 1640 supplemented with 5%FCS and 5 mg/ml PMB. PBMC were incubated at 37°C in ahumidified atmosphere containing 5% CO2 for 48 h. Aftera preincubation period of 15 min with the indicatedconcentrations of IFN-a, PBMC were stimulated eitherwith ConA, ConA/PMA, anti-CD3/anti-CD28 or anti-CD28/PMA. ConA and PMA were used in a finalconcentration of 10 mg/ml and 1 ng/ml, respectively. Theanti-CD3 and anti-CD28 antibodies were used in a solubleform at a final dilution of 1:4000 and 1:7000, respectively.IFN-a was tested in a concentration range between 0 and10 000 IU/ml.

After incubation, PBMC cultures were frozen andthawed three times and the lysates after centrifugation

stored at −70°C until cytokine detection by ELISA. Datatherefore reflect total cytokine production (secreted andcell-associated).

Figure 5. Differential effects of IFN-a on the production of IL-4 (A)and IL-13 (B) in PBMC from healthy individuals.

PBMC (2×105 cells per well) obtained from different donors werestimulated with CLB-T3/4.E anti-CD3 mAb (1:4000 dilution) andCLB-28/1 anti-CD28 mAb (1:7000 dilution) in the absence orpresence of graded concentrations of IFN-a. Controls leftunstimulated did not show any IL-4 or IL-13 synthesis. After 48 h,cells were frozen and thawed three times and the lysates were assayedfor IL-4 and IL-13 by ELISA. Mean2 SEM of five experiments areshown. *PQ 0.05 compared with 0 IU/ml IFN-a.

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Figure 6. Differential effects of IFN-a on the production of IL-4 (A)and IL-13 (B) in PBMC from healthy individuals.

PBMC (2×105 cells per well) obtained from different donors werestimulated with CLB-28/1 anti-CD28 mAb (1:7000 dilution) andPMA (1 ng/ml) in the absence or presence of graded concentrationsof IFN-a. Controls left unstimulated did not show any IL-4 or IL-13synthesis. After 48 h, cells were frozen and thawed three times andthe lysates were assayed for IL-4 and IL-13 by ELISA. Mean 2 SEMof six experiments are shown. *PQ 0.05 compared with 0 IU/mlIFN-a.

a limit of detection of 0.2 and 0.5 pg/ml, respectively, and donot show any cross-reactivity with various other cytokines.

Northern analysisPBMC (1×107) were suspended in RPMI 1640 and 5%

FCS and cultured for 5 and 24 h, respectively, afterstimulation with ConA (10 mg/ml), ConA (10 mg/ml)+PMA(1 ng/ml) (with controls left unstimulated) and addition of theindicated amounts of IFN-a 15 min in advance. The cellswere washed once with PBS (GIBCO) and then total RNAwas purified by the guanidinium-isothiocyanate phenolextraction method using the RNA-Clean system (AGSGmbH, Heidelberg, Germany) following manufacturer’sinstructions. The amount of RNA was measured byspectrophotometry at 260 nm. Ten micrograms of total RNAwas separated through 1% agarose/1.1 M formaldehyde gels,and transferred to Nytran nylon membranes (Schleicher &Schuell, Vienna, Austria) and cross-linked by short wave UVexposure in a Stratalinker (Stratagene). Filters werehybridized with probes, labelled to high specific activity bythe random primed method (Boehringer Mannheim, Vienna,Austria), washed under stringent conditions and developed.The IL-13 probe was a 842 bp Sty-I fragment of a hIL-13cDNA-insert containing plasmid kindly provided by Dr R. deWaal Malefyt, DNAX Research Institute, Palo Alto, CA. AhIL-4 insert was a gift from Prof. Dr C. Peschel, Departmentof Medicine, Johannes Gutenberg University School ofMedicine, Mainz, Germany. Control-hybridizations wereperformed with the rat cDNA of the housekeeping geneglyceraldehyde-3-phosphatedehydrogenase (GAPDH).

StatisticsData are presented as mean 2 SEM. Statistical analysis

was performed using Wilcoxon’s matched-pairs signed-ranktest. P values less than 0.05 were considered to be significant.

Acknowledgements

We gratefully acknowledge Dr Rene de WaalMalefyt for helpful discussion and providing IL-13cDNA. Supported by grant P11379 of the AustrianFund for the Promotion of Scientific Research.

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