JOURNAL OF INTERFERON & CYTOKINE RESEARCH 25:444–452 (2005)© Mary Ann Liebert, Inc.

Lack of IFN-� Production in Response to AntigenicStimulation in Human IFN-�-Treated Lymphocytes

CHRISTINE ROGEZ-KREUZ,1,2 BENJAMIN MANÉGLIER,3 NATHALIE DEREUDDRE-BOSQUET,4

DOMINIQUE DORMONT,1,5 and PASCAL CLAYETTE4

ABSTRACT

Interferon-� (IFN-�) is a type I IFN responsible for maternal recognition of the fetus in ruminants. In addi-tion to its physiologic role, IFN-� also inhibits HIV replication in human lymphocytes and macrophages anddisplays immunomodulatory effects but lacks the toxicity associated with other type I IFNs. Human IFN-�promotes a Th1 response, whereas IFN-� has anti-inflammatory properties, inducing the production of Th2cytokines in murine models of experimental autoimmune encephalitis (EAE) or fetal loss. We compared theeffects of ovine IFN-� (OvIFN-�) and human IFN-� (HuIFN-�) on cytokine mRNA and protein productionin human peripheral blood mononuclear cells (PBMCs) activated with a recall antigen, such as purified pro-tein derivative (PPD) of tuberculin or with a proinflammatory stimulus, such as lipopolysaccharide (LPS). Inboth cases, IFN-� increased IFN-� production, whereas IFN-� did not and thereby promoted Th2 cytokineproduction. This original property renders IFN-� a potential candidate for therapeutic applications in im-mune disorders, such as multiple sclerosis (MS), but its therapeutic use in the treatment of HIV infectionshould be considered with caution.

444

INTRODUCTION

TYPE I INTERFERONS (IFNS) CONSTITUTE a family of multi-functional cytokines with a broad spectrum of biologic prop-

erties.(1) They have powerful antiviral activity and play a criticalrole in modulating immune responses to foreign and self-anti-gens, being involved in the differentiation and effector functionsof natural killer (NK) cells, CD8� cytotoxic T lymphocytes(CTL), and Th cells.(2–6) Human IFN-�/� are the first cytokinesto be produced by M� and dendritic cells (DC) in response toviral infections, providing a bridge between innate and adaptiveimmunity.(7–9) Indeed, IFN-�/� have been shown to promote Th1development in human CD4� cells in vitro,(3,10) at least partlyby activating the transcription factor Stat4.(11) Th1 cells promotenot only M� activation but also the production of opsonizingcomplement-fixing antibodies (Ab) and Ab involved in Ab-de-pendent cell cytotoxicity(12,13) via production of the specific Th1cytokines, IFN-� and interleukin-2 (IL-2). Thanks to their anti-viral, antitumoral, and immunomodulatory properties, IFN-� isthe approved treatment for viral hepatitis(14) and AIDS-associ-

ated Kaposi’s sarcoma (KS)(15) and IFN-� is prescribed for mul-tiple sclerosis (MS),(16) which is considered to be an autoimmunedisease mediated by proinflammatory Th1 lymphocytes. The pu-tative mechanism of action of IFN-� includes the inhibition ofT cell proliferation, blocking of the blood-brain barrier (BBB)and T cell transmigration into the brain by interfering with celladhesion, and the upregulation of antiinflammatory Th2 cyto-kines.(17,20) Individuals with chronic viral hepatitis, however,present predominantly Th2 responses.(21) In patients respondingto IFN-� therapy, viral clearance seems to be correlated with anincrease in the Th1 response.(22–24) Moreover, whereas a Th1 toTh2 switch occurs during AIDS progression,(25,26) the ex vivotransduction of peripheral blood lymphocytes (PBL) from HIV-infected patients with a construct encoding IFN-� restores a Th1profile of cytokine production.(27) Thus, in addition to the anti-viral and antiproliferative functions of type I IFN, the effects ofthese molecules on the Th1/Th2 balance may have a major ef-fect on treatment outcome.

The trophoblast IFN-� is a member of the type I IFN fam-ily that is found exclusively in ruminant ungulate species, in

1Service de Neurovirologie, CEA, CRSSA, Université Paris XI, EPHE, IPSC, Fontenay-aux-Roses, France.2Unité de Biologie du Développement et de la Reproduction, Département de Physiologie Animale, INRA, Jouy-en-Josas, France.3Pharmacologie, Faculté de Médecine Paris Ile-de-France Ouest, Centre Hospitalier de Versailles, Le Chesnay, France.4SPI-BIO, CEA, Fontenay-aux-Roses, France.5This work is dedicated to Prof. D. Dormont, who died in November 2003.

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which it forms part of the hormonal environment required forembryonic development.(28,29) This antiluteolytic IFN, which isstructurally related to IFN-� and IFN-�, is secreted in largequantities by extraembryonic trophectoderm cells during theperi-implantation period.(30) Like other IFNs, IFN-� displaysantiviral and antiproliferative properties,(31–33) but this mole-cule is less toxic in vitro and in vivo than IFN-� or IFN-�.(34–36)

It has proved to be effective against both acute and relapsingexperimental autoimmune encephalitis (EAE), a mouse modelfor MS,(34,37) and also prevents naturally occurring fetal loss inmice.(38) In both these mouse models, IFN-� seems to act byincreasing the production of Th2 cytokines, such as IL-10, atthe expense of IFN-�.(38–40) In this study, we aimed to deter-mine the differences between IFN-� and IFN-� by comparingthe proliferative and cytokine responses to recombinant ovineIFN-� (rOvIFN-�) with those to recombinant human IFN-�2b(rHuIFN-�2b) in human antigen-stimulated memory T cells.Purified protein derivative (PPD) was used as a recall antigen.This antigen usually generates a Th1 response and strong lym-phocyte proliferation.(41,42) We used bacterial endotoxin, a pow-erful inducer of inflammation,(43) for observations of the pri-mary response.

MATERIALS AND METHODS

Cells

Human peripheral blood mononuclear cells (PBMC) wereisolated by Ficoll-Hypaque density gradient centrifugation fromthe blood of healthy donors known to be vaccinated against tu-berculosis. These cells were then dispensed into 96-well plates(150,000 cells per well) and cultured in 200 �l RPMI 1640medium supplemented with 10% heat-inactivated (�56°C for45 min) fetal bovine serum (FBS) and 1% antibiotic mixture(penicillin, streptomycin, neomycin). For antiviral assays, cellswere first activated for 48 h with 1 �g/ml phytohemagglutinin-protein (PHA-P) and then cultured in the medium described,supplemented with 20 IU/ml rHuIL-2. Cells were maintainedat 37°C in a humidified atmosphere containing 5% CO2.

Reagents

rOv-IFN-� was produced in yeast by Transgene S.A. (Stras-bourg, France), by genetic engineering techniques(44) using theIFN-� cDNA.(45) It was purified by ion-exchange HPLC. Thebatch of IFN-� and cell culture medium were shown to be en-dotoxin free by means of the limulus amebocyte lysate (LAL)test (Chromogenix, Milan, Italy). IFN-� had a specific antiviralactivity of 108 IU/mg against vesicular stomatitis virus (VSV)in Madin-Darby bovine kidney (MDBK) cells.(46) HuIFN-�2b(Schering-Plough, Kenilworth, NJ) was used as a control; it alsohad a specific antiviral activity of 108 IU/mg against VSV.

Cells were stimulated with lipopolysaccharide (LPS) (Esch-erichia coli serotype 0111: B4; Sigma, St Louis, MO), PPD(Veterinary Laboratories Agency, Surrey, U.K.), or PHA-P.

Virus and antiviral assay

PHA-P-activated PBMCs were infected with the lym-photropic reference strain HIV-1-LAI.(47) The antiviral activity

of the two IFNs was compared by quantifying the inhibition ofHIV reverse transcriptase (RT) activity in the supernatants ofIFN-treated PBMCs. IFN was added to the cultures of PBMCs24 h before infection at concentrations of 1–10,000 IU/ml. Theinfectious dose of virus used in this experiment was 500 TCID50

per 106 cells. Cell culture supernatants were collected 7 daysafter infection for quantification of RT activity with the Retro-Sys kit (Innovagen, Lund, Sweden).

Treatments and stimulations

PBMCs were treated with IFN at concentrations of 1–10,000IU/ml. To evaluate recall antigen-specific proliferation, PBMCswere subsequently stimulated with PPD at a final concentrationof 10 �g/ml. The effects of endotoxin were tested, using LPSat a final concentration of 100 ng/ml. Ten culture wells weretreated and stimulated in the same way: four were used for theevaluation of cell proliferation and six for cytokine mRNA andprotein quantification.

Proliferation assay

Treated and stimulated cells were incubated for 4 days. Cellswere then pulsed with 1 �Ci 3H-thymidine per well (specificactivity of 185 GBq/mmol) (Amersham Biosciences, ArlingtonHeights, IL). After 8 h incubation, plates were transferred to�20°C for a freezing/defrosting cycle, and DNA was precipi-tated onto glass fiber filters with an automatic cell harvester.The amount of incorporated 3H-thymidine was determined in aliquid scintillation counter and expressed as counts per min(cpm) using a Betaplate flatbed liquid scintillation counter(Perkin-Elmer Life Science, Courtaboeuf, France).

Real-time PCR for cytokine mRNA quantification

Treated and stimulated cells were incubated for 3 days. Onday 3, cells were harvested, and supernatants were collected forcytokine quantification by ELISA. Cells were lysed, and totalRNA was extracted with the RNeasy Mini Kit (Qiagen Sciences,Germantown, MD), according to the manufacturer’s instructions.The total RNA preparation was treated with RNase-free DNase(Qiagen Sciences) and subjected to reverse transcription witholigo-dT primers. Real-time PCR was performed with an iCy-cler iQ Real-Time PCR Detection System (Bio-Rad, Hercules,CA). Specific primers were used for HuIFN-� (sense: 5�-GGTTCT CTT GGC TGT TAC TGC-3�; antisense: 5�-CCT TTT TCGCTT CCC TGT T-3�), IL-10 (sense: 5�-CCA AGC TGA GAACCA AGA CC-3�; antisense: 5�-CAC TCA TGG CTT TGTAGA CGC-3�), IL-2 (sense: 5�-ACC TCA ACT CCT GCC ACAAT; antisense: 5�-GCC TTC TTG GGC ATG TAA AA-3�), IL-4 (sense: 5�-ACT GTG CTC CGG CAG TTC TAC-3�; antisense:5�-GGT TCC TGT CGA GCC GTT TCA-3�), and the house-keeping gene GAPDH (sense: 5�-TCG TGG AAG GAC TCATGA CC-3�; antisense: 5�-TCA GCT CAG GGA TGA CCT TG-3�). PCR was performed in a reaction volume of 25 �l PCR iQSYBR Green supermix (Bio-Rad) with 0.4 �M of each primer.The thermal profile comprised initial denaturation (95°C for 2min) and 40 amplification cycles (95°C for 15 sec, 58°C for 50sec, and 72°C for 20 sec), followed by melting curve analysis toassess the specificity of amplification. Results are given as ratioswith respect to GAPDH.

OVINE IFN-� INDUCES Th2 BIAS IN HUMAN LYMPHOCYTES 445

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ELISA for cytokine measurement

Cytokine production was quantified in cell culture super-natants, using Quantikine immunoassay kits according to themanufacturer’s instructions (R&D Systems Europe, Abingdon,U.K.).

Data analysis

Each experiment was performed with cells obtained from theperipheral blood of a single donor, and the procedure was thenrepeated for 5 other donors. There were interindividual varia-tions in the levels of thymidine incorporation by PBMCs in re-sponse to specific stimuli and in the amounts of cytokine pro-duced, although all gave the same pattern of response to eachof the stimuli. For clarity, the results of one complete series ofexperiments using responder PBMCs from one blood donor arepresented in each figure. The data obtained from each blooddonor were analyzed, using Student’s unpaired t-test (StatviewF 4.5, SAS Institute Inc., Cary, NC). Differences were consid-ered to be significant if p � 0.01 (**) or p � 0.05 (*) and non-significant (n.s.) if p � 0.05 and if the results were reproduciblefor all blood donors tested.

RESULTS

Comparison of antiviral and antiproliferative activitiesof HuIFN-� and OvIFN-� in human PBMCs

Both IFN-� and IFN-� displayed strong antiretroviral activ-ity in human PHA-P-activated PBMCs. The activity of HIV RTmeasured in the supernatants of PBMCs 7 days after infectionwas significantly decreased in a dose-dependent manner by type I IFN (Fig. 1A). IFN-� seemed to have slightly stronger anti-retroviral activity than IFN-�, but this difference was signifi-cant only at the highest concentration.

In parallel, we compared the antiproliferative effects of bothtypes of IFN in PHA-P-stimulated PBMCs (Fig. 1B) and PPD-stimulated PBMCs (Fig. 1C). In PHA-P-stimulated PBMCs,IFN-� had a significant and dose-dependent antilymphoprolif-erative effect from low concentrations, whereas IFN-� inhib-ited lymphocyte proliferation only at concentrations of 1,000and 10,000 IU/ml (Fig. 1B). Differences between IFN-� andIFN-� were statistically significant at every concentration testedwith PHA-P stimulation.

Thus, in PHA-P-stimulated PBMCs, the antiretroviral effectsof the IFN were similar, but IFN-� had a weaker effect than

ROGEZ-KRUEZ ET AL.446

FIG. 1. Antiretroviral (A) and antilymphoproliferative(B and C) effects of IFN-� and IFN-�. (A) RT activity in supernatants of PHA-P-stimulated 7-day-infectedPBMCs. Results are expressed as means � standard de-viation (SD) for seven culture wells. (B) Cell prolifera-tion in response to a stimulation with PHA-P for 4 days.Results are expressed as means � SD for four cultures.(C) Cell proliferation in response to stimulation with PPDfor 4 days. Results are expressed as means � SD for fourculture wells. Results correspond to a single blood donorbut are representative of experiments performed on 6 dif-ferent blood donors. Significant differences between IFN-� and IFN-� are indicated by **p � 0.01 or *p � 0.05.

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IFN-� on human lymphocyte proliferation, suggesting thatthese two properties do not involve the use of a common sig-naling pathway. The same result could be observed in PPD-stimulated PBMCs, using cells from individuals known to havebeen immunized with PPD (Fig. 1C). The effects of IFN-� andIFN-� on cell proliferation in response to PPD stimulation dif-fered significantly at all concentrations except the lowest tested.

Although IFN-� and IFN-� had similar retroviral activities,they differed in terms of antiproliferative efficiency regardless

of the stimulation used. We, therefore, focused on the cytokineenvironment induced by these two IFNs in PPD-stimulated orLPS-stimulated PBMCs.

Th1 and Th2 cytokine mRNA profiles in IFN-treatedand PPD-stimulated PBMCs

Stimulation with 10 �g/ml PPD (Fig. 2A) induced high ratesof proliferation. As previously observed, the antiproliferative

OVINE IFN-� INDUCES Th2 BIAS IN HUMAN LYMPHOCYTES 447

FIG. 2. Cell proliferation and cytokine mRNA modulations in PPD-stimulated PBMCs in response to IFN-� and IFN-�. (A)Cell proliferation in response to stimulation with PPD for 4 days. Results are expressed as means � SD for four culture wells.IFN-� (B), IL-2 (C), IL-10 (D), and IL-4 (E) mRNA levels in PBMCs stimulated with PPD for 3 days and treated with IFN-�or IFN-�. Results correspond to a single blood donor but are representative of experiments performed on 6 different blood donors.Significant differences between IFN-� and IFN-� are indicated by **p � 0.01 or *p � 0.05.

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effects of the two IFNs differed significantly at the three con-centrations tested. We quantified the levels of mRNA for Th1(IFN-� and IL-2) and Th2 (IL-10 and IL-4) cytokines in theseIFN-treated PBMCs stimulated with PPD for 3 days.

As expected, the PPD stimulation of untreated cells increasedIFN-� mRNA production (Fig. 2B) but had no significant im-pact on IL-2 mRNA production (Fig. 2C). In contrast, themRNA levels of Th2 cytokines, IL-10 and IL-4, were stronglydecreased by PPD stimulation (Fig. 2D,E).

The dose-dependent effects of IFN-� and IFN-� on PPD-induced cytokine profiles were then compared. The two lowest IFN concentrations did not affect IFN-� mRNA lev-els. In contrast, whereas 10,000 IU/ml IFN-� decreased IFN-� mRNA levels, the same dose of IFN-� significantly in-creased them (Fig. 2B). IL-2 mRNA levels were increased ina dose-dependent manner by IFN-� but were significantly in-creased by IFN-� only at a concentration of 10,000 IU/ml(Fig. 2C).

Interestingly, both IFNs inhibited the PPD-induced decreasein IL-10 mRNA levels. A concentration of 100 IU/ml IFN-�was sufficient to restore IL-10 mRNA levels in unstimulatedcells, and a similar effect was observed with the two highestconcentrations of IFN-� (Fig. 2D). At a concentration of 1000IU/ml, IFN-� even increased the basal level of IL-10 mRNA(Fig. 2D). IL-4 mRNA production was unaffected by IFN-� butwas slightly increased by IFN-� at a concentration of 1000IU/ml (Fig. 2E). However, this effect was not dose dependentand was insufficient to counteract the PPD-induced decrease.

These results deal with the effects of IFN on mRNA pro-duction for Th1 and Th2 cytokines in response to a recall anti-gen inducing a Th1 bias. We also evaluated the effects of IFNin PBMCs stimulated with bacterial endotoxins, which are pow-erful inducers of fever and inflammation.

Th1 and Th2 cytokine mRNA profiles in IFN-treatedand LPS-stimulated PBMCs

Stimulation with 100 ng/ml LPS induced cell proliferation,but to a lesser extent than PPD (Fig. 3A). In contrast to the re-sults obtained with PPD stimulation, the antiproliferative ef-fects of IFN-� were significant only at a concentration of 10,000IU/ml (Fig. 3A). In parallel, IFN-� did not significantly modu-late cell proliferation at the concentration tested. After 3 daysof stimulation, quantification of mRNA for Th1 and Th2 cytokines in PBMCs showed an increase in mRNA levels forIFN-� (Fig. 3B) and IL-10 (Fig. 3D), a decrease in mRNA levels for IL-2 (Fig. 3C), and no change in mRNA levels forIL-4 (Fig. 3E).

The levels of mRNA for Th1 cytokines were generally higherin IFN-�-treated PBMCs, whereas those for Th2 cytokines weregenerally higher in IFN-�-treated PBMCs. Indeed, the LPS-in-duced increase in IFN-� mRNA levels was increased by IFN-�in a dose-dependent manner (Fig. 3A). In contrast, IFN-� onlyslightly increased IFN-� mRNA levels at a concentration of10,000 IU/ml (Fig. 3A). The LPS-induced decrease in IL-2mRNA levels was counteracted by 100 IU/ml IFN-�, as the lev-els of this mRNA were higher than those in unstimulated cells(Fig. 3C). Higher doses of IFN-� or of IFN-� for the concen-trations tested caused IL-2 mRNA levels similar to those in un-stimulated PBMCs (Fig. 3C). IL-10 mRNA levels were in-

creased by IFN-� at all three concentrations used (Fig. 3D).However, this effect was most marked for 100 IU/ml IFN-�(Fig. 3D). Although IL-10 mRNA levels remained significantlyhigher than those of the untreated control, IFN-� decreased IL-10 mRNA levels in a dose-dependent manner (Fig. 3D).IFN-� treatment gave a similar IL-10 mRNA profile, althoughthe level of induction observed at a concentration of 100 IU/mlwas lower than that with IFN-� and remained not significant.A dose-dependent decrease in IL-10 mRNA level was observedwith IFN-� (Fig. 3D). In parallel, levels of mRNA for the Th2cytokine IL-4 were reduced to those of unstimulated cells byIFN-� at every concentration tested, whereas this was the caseonly for the highest concentration of IFN-� tested (Fig. 3E).

Production of Th1 and Th2 cytokines in supernatantsof IFN-treated, LPS-stimulated PBMCs

As the levels of mRNA for IFN-� and IL-10, the major Th1and Th2 cytokines, respectively, were the most strongly affectedby IFN, we evaluated the effects of IFN on the production ofthese cytokines in the supernatants of unstimulated, PPD-stim-ulated, or LPS-stimulated PBMCs. The effect of IFN alone was first studied in unstimulated PBMCs and is shown in Fig-ure 4A. Few significant effects were observed, and although IFN-� slightly increased IL-10 production, this dose-dependentincrease was not significant. The amounts of IL-10 in the su-pernatants of IFN-�-treated PBMCs were smaller than those inthe supernatants of IFN-�-treated cells. In contrast, IFN-� in-creased IFN-� production when used at a concentration of10,000 IU/ml, whereas IFN-� had no effect.

PPD stimulation significantly upregulated the synthesis ofIL-10 and IFN-� (Fig. 4B). In contrast to what was observedat the mRNA level, however, we observed no significantchange in the level of IL-10 protein in response to IFN-� af-ter 3 days of stimulation. In contrast to what we observed forIL-10 mRNA, IL-10 protein levels in PBMC culture super-natants were decreased by IFN-� in a dose-dependent manner.As observed in unstimulated cells and consistent with the results obtained for IFN-� mRNA, IFN-� protein levels wereunaffected in IFN-treated PBMCs, except for 10,000 IU/mlIFN-� (Fig. 4B).

In the supernatants of LPS-stimulated cells (Fig. 4C), cyto-kine levels were much higher than those in unstimulated cells.As observed with PPD stimulation, IL-10 levels were unaf-fected by IFN-� but were decreased in a dose-dependent man-ner by IFN-�. Conversely, IFN-� production was strongly increased by IFN-� and only slightly increased by high con-centrations of IFN-�.

DISCUSSION

Type I IFN molecules are of potential therapeutic value, forboth their antiviral functions and their immunomodulatoryproperties. They are used in the treatment of viral infections,such as hepatitis,(14) and in the treatment of diseases caused byimmune imbalance, such as MS.(16) This comparison of the im-munomodulatory effects of HuIFN-� and OvIFN-� in humanstimulated PBMCs revealed a major difference between thesetwo type I IFNs. IFN-� increases the production of Th1 cyto-

ROGEZ-KRUEZ ET AL.448

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kines, whereas the level of production of Th2 cytokines is consistently higher in IFN-�-treated PBMCs, suggesting that IFN-� promotes a Th1 lymphocyte phenotype and IFN-� doesnot.

IFN-� has been reported to induce IL-10 synthesis in mousemodels of fetal loss(38) and EAE.(34,48) Soos et al.(40) also dem-onstrated that, unlike IFN-�/�, IFN-� cannot induce IFN-� se-cretion in vivo in myelin basic protein-specific T cell receptor(TCR)-transgenic mice, whereas the levels of IL-10 secreted in

response to this IFN are similar to those obtained with IFN-�/�. In contrast, IFN-� has been shown to promote Th1 cyto-kine production in IFN-treated individuals.(21,49) Some of thebeneficial and adverse effects of IFN-� are thought to resultfrom the promotion of a Th1 type of immune response by thiscytokine.(2)

PPD stimulation is known to induce lymphocyte prolifera-tion and a Th1 orientation(50) by increasing IFN-� lymphocytesecretion. In this study, 3 days of stimulation with PPD in-

OVINE IFN-� INDUCES Th2 BIAS IN HUMAN LYMPHOCYTES 449

FIG. 3. Cell proliferation and cytokine mRNA modulations in LPS-stimulated PBMCs in response to IFN-� and IFN-�. (A)Cell proliferation in response to stimulation with LPS for 4 days. Results are expressed as means � SD for four culture wells.IFN-� (B), IL-2 (C), IL-10 (D), and IL-4 (E) mRNA levels in PBMCs stimulated for 3 days with LPS and treated with IFN-�or IFN-�. Results correspond to a single blood donor but are representative of experiments performed on six different blooddonors. Significant differences between IFN-� and IFN-� are indicated by **p � 0.01 or *p � 0.05.

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creased the production of both mRNA and protein for IFN-�. Paradoxically, whereas IL-10 mRNA levels decreased inresponse to PPD, IL-10 protein levels increased in cell cul-ture supernatants, suggesting negative feedback modulatingIL-10 secretion.(51) In this stimulation context, IFN-� en-hanced the PPD-induced Th1 cytokine environment. Al-though IL-10 mRNA levels were also increased by IFN-�,

we detected a dose-dependent decrease in IL-10 protein se-cretion after 3 days of treatment. In contrast, IFN-� did notincrease IFN-� production but counteracted the PPD-induceddecrease in IL-10 mRNA levels, thereby affecting the PPD-induced Th1 bias.

Bacterial endotoxins are nonspecific inducers of inflamma-tion. Stimulation with these molecules increased the release of

ROGEZ-KRUEZ ET AL.450

FIG. 4. Cytokine synthesis in unstimulated (A), PPD-stimulated (B), and LPS-stimulated (C) PBMCs in response to IFN-� andIFN-�. Results are expressed as means � SD for four culture wells and correspond to a single blood donor but are representa-tive of experiments performed on six different blood donors. Significant differences between IFN-� and IFN-� are indicated by**p � 0.01 or *p � 0.05.

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both Th1 and Th2 cytokines by PBMCs. In this context, the an-tiproliferative effects of type I IFN were less marked, and theeffects of IFN-� and IFN-� on cytokine secretion differed sig-nificantly. IFN-� induced high levels of IFN-� secretion anddecreased IL-10 synthesis, thereby promoting the inflammatoryresponse. In contrast, IFN-� increased IL-10 mRNA levels, butthe corresponding protein levels did not increase after 3 daysof treatment. Furthermore, IFN-� did not increase IFN-� pro-duction. Our results confirm the findings of other studies onIFN-� in mouse models; this cytokine seems to have anti-in-flammatory properties similar to those of IFN-� in the treat-ment of MS.(17)

In the pathologic context of HIV infection, a Th1 to Th2switch has been reported in patients,(25,52) correlated with dis-ease progression. Therefore, immunotherapy based on cyto-kines or cytokine antagonists has been considered for the treat-ment of HIV disease. Indeed, Th1 cytokines, such as IFN-� andIL-12, or antagonizing Th2 cytokines, such as IL-4 and IL-10,may stimulate lymphopoiesis and enhance cell-mediated im-munity, restoring the cytokine balance.(53) Thus, despite its an-tiretroviral properties(32,35) and its low toxicity, IFN-� may notbe appropriate for the treatment of HIV infection. However, itis still of potential value for the treatment of immune disorders,such as MS, as efficient treatment promotes the production ofTh2 cytokines at the expense of IFN-�.(54) Moreover, a phaseI study of the safety and biologic effects of IFN-� in the treat-ment of MS showed no clinically significant toxicity,(55) andthis compound, therefore, has considerable potential for thera-peutic application in this disease.

ACKNOWLEDGMENTS

We thank Julie Sappa for excellent help in preparation of themanuscript, Dr. Jacques Martal from the Institut National de laRecherche Agronomique (INRA), and the Centre de Transfu-sion Sanguine des Armées (CTSA, Hôpital Percy, Clamart,France). This work was supported by Ensemble contre le SIDA-SIDACTION, the Commissariat à l’Energie Atomique (CEA),and the INRA.

REFERENCES

1. Stark GR, Kerr IM, Williams BR, Silverman RH, Schreiber RD.How cells respond to interferons. Annu. Rev. Biochem. 1998;67:227–264.

2. Belardelli F, Gresser I. The neglected role of type I interferon inthe T-cell response: implications for its clinical use. Immunol. To-day 1996;17:369–372.

3. Parronchi P, De Carli M, Manetti R, Simonelli C, Sampognaro S,Piccinni MP, Macchia D, Maggi E, Del Prete G, Romagnani S. IL-4 and IFN (alpha and gamma) exert opposite regulatory effects onthe development of cytolytic potential by Th1 or Th2 human T cellclones. J. Immunol. 1992;149:2977–2983.

4. Parronchi P, Mohapatra S, Sampognaro S, Giannarini L, Wahn U,Chong P, Mohapatra S, Maggi E, Renz H, Romagnani S. Effectsof interferon-alpha on cytokine profile, T cell receptor repertoireand peptide reactivity of human allergen-specific T cells. Eur. J.Immunol. 1996;26:697–703.

5. Hunter CA, Gabriel KE, Radzanowski T, Neyer LE, RemingtonJS. Type I interferons enhance production of IFN-gamma by NKcells. Immunol. Lett. 1997;59:1–5.

6. Biron CA. Activation and function of natural killer cell responsesduring viral infections. Curr. Opin. Immunol. 1997;9:24–34.

7. Le Bon A, Tough DF. Links between innate and adaptive im-munity via type I interferon. Curr. Opin. Immunol. 2002;14:432–436.

8. Santini SM, Di Pucchio T, Lapenta C, Parlato S, Logozzi M, Be-lardelli F. The natural alliance between type I interferon and den-dritic cells and its role in linking innate and adaptive immunity. J.Interferon Cytokine Res. 2002;22:1071–1080.

9. Kadowaki N, Antonenko S, Lau JY, Liu YJ. Natural interferon al-pha/beta-producing cells link innate and adaptive immunity. J. Exp.Med. 2000;192:219–226.

10. Brinkmann V, Geiger T, Alkan S, Heusser CH. Interferon alphaincreases the frequency of interferon gamma-producing humanCD4� T cells. J. Exp. Med. 1993;178:1655–1663.

11. Rogge L, D’Ambrosio D, Biffi M, Penna G, Minetti LJ, PreskyDH, Adorini L, Sinigaglia F. The role of Stat4 in species-specificregulation of Th cell development by type I IFNs. J. Immunol.1998;161:6567–6574.

12. Abbas AK, Murphy KM, Sher A. Functional diversity of helper Tlymphocytes. Nature 1996;383:787–793.

13. Romagnani S. Understanding the role of Th1/Th2 cells in infec-tion. Trends Microbiol. 1996;4:470–473.

14. Cacoub P, Benhamou Y. [Role of interferons in the treatment ofhepatitis B and hepatitis C virus infections.] Rev. Med. Interne2002;23(Suppl 4):459s–474s.

15. Krown SE. IFN-�: evolving therapy for AIDS-associated Kaposi’ssarcoma. J. Interferon Cytokine Res. 1998;18:209–214.

16. Coyle PK, Hartung HP. Use of interferon beta in multiple sclero-sis: rationale for early treatment and evidence for dose- and fre-quency-dependent effects on clinical response. Mult. Scler.2002;8:2–9.

17. Kozovska ME, Hong J, Zang YC, Li S, Rivera VM, Killian JM,Zhang JZ. Interferon beta induces T-helper 2 immune deviation inMS. Neurology 1999;53:1692–1697.

18. Rep MH, Schrijver HM, van Lopik T, Hintzen RQ, Roos MT, AderHJ, Polman CH, van Lier RA. Interferon (IFN)-beta treatment en-hances CD95 and interleukin 10 expression but reduces interferon-gamma producing T cells in MS patients. J. Neuroimmunol.1999;96:92–100.

19. Huang YM, Stoyanova N, Jin YP, Teleshova N, Hussien Y, XiaoB, Fredrikson S, Link H. Altered phenotype and function of blooddendritic cells in multiple sclerosis are modulated by IFN-beta andIL-10. Clin. Exp. Immunol. 2001;124:306–314.

20. Furlan R, Bergami A, Lang R, Brambilla E, Franciotta D, Mar-tinelli V, Comi G, Panina P, Martino G. Interferon-beta treatmentin multiple sclerosis patients decreases the number of circulatingT cells producing interferon-gamma and interleukin-4. J. Neu-roimmunol. 2000;111:86–92.

21. Xing T, Zhang L, Lu Q, Hou J, Feng X, Luo K. Th1/Th2 type cy-tokines in hepatitis B patients treated with interferon-alpha. Chin.Med J. (Engl.) 2001;114:921–924.

22. Shinohara M, Ishii K, Takamura N. Long-term changes of periph-eral blood CD4-positive T cell subsets (Th1, Th2) in chronic he-patitis C patients with a sustained response or no response to IFN.Hepatol. Res. 2003;27:260–265.

23. Amati L, Caradonna L, Magrone T, Mastronardi ML, Cuppone R,Cozzolongo R, Manghisi OG, Caccavo D, Amoroso A, Jirillo E.Modifications of the immune responsiveness in patients with he-patitis C virus infection following treatment with IFN-alpha/rib-avirin. Curr. Pharm. Des. 2002;8:981–993.

24. Cacciarelli TV, Martinez OM, Gish RG, Villanueva JC, Krams SM.Immunoregulatory cytokines in chronic hepatitis C virus infection:pre- and posttreatment with interferon alfa. Hepatology 1996;24:6–9.

25. Clerici M, Shearer GM. A Th1 � Th2 switch is a critical step inthe etiology of HIV infection. Immunol. Today 1993;14;107–111.

OVINE IFN-� INDUCES Th2 BIAS IN HUMAN LYMPHOCYTES 451

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/13.

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onl

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26. Romagnani S, Maggi E. Th1 versus Th2 responses in AIDS. Curr.Opin. Immunol. 1994;6:616–622.

27. Vieillard V, Cremer I, Lauret E, Rozenbaum W, Debre P, AutranB, De Maeyer E. Interferon beta transduction of peripheral bloodlymphocytes from HIV-infected donors increases Th1-type cyto-kine production and improves the proliferative response to recallantigens. Proc. Natl. Acad. Sci. USA 1997;94:11595–11600.

28. Liu L, Leaman DW, Bixby JA, Roberts RM. A type I ovine inter-feron with limited similarity to IFN-alpha, IFN-omega and IFN-tau: gene structure, biological properties and unusual species speci-ficity. Biochim. Biophys. Acta 1996;1294:55–62.

29. Martal JL, Chene NM, Huynh LP, L’Haridon RM, Reinaud PB,Guillomot MW, Charlier MA, Charpigny SY. IFN-tau: a novel sub-type I IFN1. Structural characteristics, non-ubiquitous expression,structure-function relationships, a pregnancy hormonal embryonicsignal and cross-species therapeutic potentialities. Biochimie1998;80:755–777.

30. Roberts RM. IFN-� and pregnancy. J. Interferon Cytokine Res.1996;16:271–273.

31. Pontzer CH, Bazer FW, Johnson HM. Antiproliferative activity ofa pregnancy recognition hormone, ovine trophoblast protein-1.Cancer Res. 1991;51:5304–5307.

32. Rogez C, Martin M, Dereuddre-Bosquet N, Martal J, Dormont D,Clayette P. Anti-human immunodeficiency virus activity of tau in-terferon in human macrophages: involvement of cellular factorsand beta-chemokines. J. Virol. 2003;77:12914–12920.

33. Pontzer CH, Torres BA, Vallet JL, Bazer FW, Johnson HM. Anti-viral activity of the pregnancy recognition hormone ovine trophoblastprotein-1. Biochem. Biophys. Res. Commun. 1988;152:801–807.

34. Soos JM, Subramaniam PS, Hobeika AC, Schiffenbauer J, John-son HM. The IFN pregnancy recognition hormone IFN-tau blocksboth development and superantigen reactivation of experimentalallergic encephalomyelitis without associated toxicity. J. Immunol.1995;155:2747–2753.

35. Dereuddre-Bosquet N, Clayette P, Martin M, Mabondzo A, FretierP, Gras G, Martal J, Dormont D. Anti-HIV potential of a new in-terferon, interferon-tau (trophoblastin). J. Acquir. Immune Defic.Syndr. Hum. Retrovirol. 1996;11:241–246.

36. Subramaniam PS, Khan SA, Pontzer CH, Johnson HM. Differen-tial recognition of the type I interferon receptor by interferons tauand alpha is responsible for their disparate cytotoxicities. Proc.Natl. Acad. Sci. USA 1995;92:12270–12274.

37. Mujtaba MG, Streit WJ, Johnson HM. IFN-tau suppresses both theautoreactive humoral and cellular immune responses and inducesstable remission in mice with chronic experimental allergic en-cephalomyelitis. Cell. Immunol. 1998;186:94–102.

38. Chaouat G, Assal Meliani A, Martal J, Raghupathy R, Elliot J, Mos-mann T, Wegmann TG. IL-10 prevents naturally occurring fetal lossin the CBA DBA/2 mating combination, and local defect in IL-10production in this abortion-prone combination is corrected by in vivoinjection of IFN-tau. J. Immunol. 1995;154:4261–4268.

39. Mujtaba MG, Soos JM, Johnson HM. CD4 T suppressor cells me-diate interferon tau protection against experimental allergic en-cephalomyelitis. J. Neuroimmunol. 1997;75:35–42.

40. Soos JM, Stuve O, Youssef S, Bravo M, Johnson HM, Weiner HL,Zamvil SS. Cutting edge: oral type I IFN-tau promotes a Th2 biasand enhances suppression of autoimmune encephalomyelitis byoral glatiramer acetate. J. Immunol. 2002;169:2231–2235.

41. Henderson DC, Rippin JJ. Stimulus-dependent production of cy-tokines and pterins by peripheral blood mononuclear cells. Im-munol. Lett. 1995;45:29–34.

42. Skoberne M, Malovrh T, Skralovnik-Stern A, Kotnik V. Humanperipheral blood lymphocytes sensitised to PPD respond to in vitrostimulation with increased expression of CD69 and CD134 acti-vation antigens and production of Th1- type cytokines. PflugersArch. 2000;440(Suppl. 5):R58–60.

43. Morrison DC, Silverstein R, Luchi M, Shnyra A. Structure-func-tion relationships of bacterial endotoxins. Contribution to micro-bial sepsis. Infect. Dis. Clin. North Am. 1999;13:313–340.

44. Degryse E, Dietrich M, Nguyen M, Achstetter T, Charlier M,Charpigny G, Gaye P, Martal J. Addition of a dipeptide spacer sig-nificantly improves secretion of ovine trophoblast interferon inyeast. Gene 1992;118:47–53.

45. Charlier M, Hue D, Boisnard M, Martal J, Gaye P. Cloning andstructural analysis of two distinct families of ovine interferon-�genes encoding functional class II and trophoblast (oTP) �-inter-feron. Mol. Cell. Endocrinol. 1991;76:161–171.

46. L’Haridon RM, Huynh L, Assal NE, Martal J. A single intrauter-ine infusion of sustained recombinant ovine interferon-tau extendscorpus luteum lifespan in cyclic ewes. Theriogenology 1995;43:1031–1045.

47. Barre-Sinoussi F, Chermann JC, Rey F, Nugeyre MT, Chamaret S,Gruest J, Dauguet C, Axler-Blin C, Vezinet-Brun F, Rouzioux C,Rozenbaum W, Montagnier L. Isolation of a T-lymphotropic retro-virus from a patient at risk for acquired immune deficiency syn-drome (AIDS). Science 1983;220:868–871.

48. Soos JM, Mujtaba MG, Subramaniam PS, Streit WJ, Johnson HM.Oral feeding of interferon tau can prevent the acute and chronic re-lapsing forms of experimental allergic encephalomyelitis. J. Neu-roimmunol. 1997;75:43–50.

49. Hempel G, Galle PR, Lohr HF. Quantitative analysis of specificTh1/Th2 helper cell responses and IgG subtype antibodies in in-terferon-alpha-treated patients with chronic hepatitis C. J. Med. Vi-rol. 2001;64:340–349.

50. Del Prete GF, De Carli M, Mastromauro C, Biagiotti R, MacchiaD, Falagiani P, Ricci M, Romagnani S. Purified protein derivativeof Mycobacterium tuberculosis and excretory-secretory antigen(s)of Toxocara canis expand in vitro human T cells with stable andopposite (type 1 T helper or type 2 T helper) profile of cytokineproduction. J. Clin. Invest. 1991;88:346–350.

51. Knolle PA, Uhrig A, Protzer U, Trippler M, Duchmann R, MeyerZM, Buschenfelde KH, Gerken G. Interleukin-10 expression is au-toregulated at the transcriptional level in human and murine Kupf-fer cells. Hepatology 1998;27:93–99.

52. Barcellini W, Rizzardi GP, Borghi MO, Fain C, Lazzarin A, MeroniPL. Th1 and Th2 cytokine production by peripheral blood mononu-clear cells from HIV-infected patients. AIDS 1994;8:757–762.

53. Galanaud P, Fior R, Boue F, Emilie D. Immunointervention basedon Th2-type cytokines in HIV infection. Eur. Cytokine Netw.1997;8:315–316.

54. Jansson A, Ernerudh J, Kvarnstrom M, Ekerfelt C, Vrethem M.Elispot assay detection of cytokine secretion in multiple sclerosispatients treated with interferon-beta1a or glatiramer acetate com-pared with untreated patients. Mult. Scler. 2003;9:440–445.

55. Olek MJ, Smith DR, Cook SL, Khoury SJ, Weiner HL. Phase Istudy of oral recombinant ovine interferon-tau in relapsing-remit-ting multiple sclerosis. Neurology 2001;56(Suppl. 3):A76.

Address reprint requests or correspondence to:Dr. Pascal ClayetteSPIBIO/DSV/DRM

Commissariat à l’Energie Atomique18 route du Panorama B.P. 6

92265 Fontenay-aux-Roses cedexFrance

Tel: �33 1 46 54 87 69Fax: �33 1 46 54 77 26

E-mail: pascal.clayette@cea.fr

Received 12 November 2004/Accepted 26 January 2005

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