doi:10.1006/cyto.1999.0561, available online at http://www.idealibrary.com on

ESSENTIAL PATHOGENETIC ROLE FORINTERFERON (IFN-)� IN CONCANAVALIN

A-INDUCED T CELL-DEPENDENT HEPATITIS:EXACERBATION BY EXOGENOUS IFN-� AND

PREVENTION BY IFN-�RECEPTOR-IMMUNOGLOBULIN FUSION

PROTEIN

Ferdinando Nicoletti,1 Paola Zaccone,2 Ming Xiang,2 Gaetano Magro,3

Maurizio Di Mauro,4 Roberto Di Marco,2 Gianni Garotta,5 PierLuigi Meroni6

We have studied the effects of either exogenously-administered interferon (IFN-)� or of anonimmunogenic mouse IFN-� receptor-Immunoglobulin (IFN-� R-Ig) fusion protein on thedevelopment of Concanavalin (Con)A-induced hepatitis in NMRI mice. PBS-treated controlmice injected with 20 mg/kg ConA developed classical serological and histological signs ofhepatitis with elevation of transaminases in the blood and infiltration of the liver by mononuclearcells and neutrophils. Treating the mice with rat IFN-� 24 h prior to and 1 h after ConA-challenge markedly exacerbated these signs of hepatitis in a dose-dependent fashion. Moreover,mice injected with lower, non hepatitogenic, doses of ConA (10, 5 mg/kg) became fullysusceptible to develop hepatitis upon similar treatment with IFN-�. Concordantly, ConA-inducedhepatitis was abrogated by either IFN-� R-Ig fusion protein or anti-IFN-� mAb. These dataprovide further evidence for the central pathogenetic role of endogenous IFN-� in ConA-inducedhepatitis and demonstrate the feasibility to prevent disease development by means of a nonimmunogenic IFN-� R-Ig fusion protein.

� 2000 Academic Press

From the 1Institute of Microbiology, University of Milan, Italy;2Department of Microbiological and Gynecological Sciences,3Institute of Anatomopathology, 4Institute of Clinical Medicine,University of Catania; Italy; 5Ares Serono, Geneve, Switzerland;6IRCCS Hospital Auxolofico, Milan, Italy

Correspondence to: Ferdinando Nicoletti, MD, Via Luigi Sturznon.3, 95021, Cannizzaro, Catania, Italy; E-mail:ferdinic@ctonline.it

Received 12 February 1999; accepted for publication 4 June 1999� 2000 Academic Press1043–4666/00/040315+09 $35.00/0

Interferon (IFN-)� is a prototypical type 1cytokine produced by T lymphocytes and NaturalKiller (NK) cells that has pleiotropic effects on theimmune system, including activation of macrophages,cytotoxic T cells and NK cells, regulation of antibody(Ab) production by B lymphocytes and control ofapoptosis (see1,2 for reviews). IFN-� induces polariz-ation of Th0 cells to a Th1 cytokine secretory pheno-type with production of interleukin (IL)-2, and tumournecrosis factor (TNF), and it also up-regulates its ownproduction in an autocrine fashion. Contempor-aneously, IFN-� down-regulates Th2 responses by

CYTOKINE, Vol. 12, No. 4 (April), 2000: pp 315–323

inhibiting the action/production of type 2 cytokinessuch as IL-4 and IL-10 that favours differentiation ofTh0 cells into the Th2 subset.1,2

Owing to its capacity to up-regulate the functionof cell mediated immune effectors, a pathogenetic rolehas been envisaged for IFN-� in organ-specific auto-immune diseases where delayed type hypersensitivity(DTH)-like responses against relevant autoantigensare involved such as autoimmune thyroiditis, type 1diabetes mellitus, multiple sclerosis, orchitis andautoimmune hepatitis.1–10

Recently, a new model of immunoinflammatoryhepatitis has been described that can be provoked inmice by one single injection with Concanavalin(Con)A.11–13 The disease is characterized by inflam-matory infiltration of the liver by neutrophils, macro-phages and T cells that occurs with a marked andsimultaneous increase in levels of transaminases in theblood shortly (8–24 h) after ConA challenge.11–13 Ithas been proposed that ConA-injection provokesmigration of splenic T cells to the liver where theycause apoptotic cell death and necrosis of hepatocytes

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mediated by perforin/granzymes and activation ofmacrophages.11–15 It has also been shown that ConA-induced hepatitis depends on a finely regulated balancebetween pro-inflammatory (IL-4, IL-12, TNF-�,IFN-�) and anti-inflammatory (IL-6, IL-10) cytokinesthat appear with different kinetics in the circulation ofConA-injected mice.8–19 ConA-induced hepatitis ismacrophage/T cell dependent as it cannot be inducedin nude athimic mice and is prevented by immunosup-pressive drugs targeting T cells and macrophages suchas Cyclosporin A, FK50611 and sodium fusidate19 orby silica particles which block macrophage functions.11

The key pathogenetic role of endogenous IFN-� inthis experimental model has been confirmed in inde-pendent studies by means of neutralizing Abs8,9 andexplicitly proved by the resistance to the disease oftransgenic mice expressing a null form of IFN-�.10

Nonetheless, several questions remain unansweredregarding both the possible clinical application of thesedata (anti-IFN-� Ab are often immunogenic and diffi-cult to use in humans20–22) and the understanding of ifand how blockage of endogenous IFN-� with Abs orits genetic deficiency in mice influences the ConA-induced cytokine release that accompanies, and prob-ably mediates, the hepatitogenic properties of ConA.Moreover, the impact of IFN-� treatment on thecourse of ConA-induced hepatitis has not been inves-tigated. This should have been important as exogenousIFN-� might act in a different and often oppositefashion than the endogenous IFN-�.5,23 To answerthese clinical and pathogenetic questions this studyinvestigated the effects of non-immunogenic murineIFN-� receptor-Immunoglobun (IFN-� R-Ig) fusionprotein receptor as a potential alternative to neutraliz-ing Abs in antagonizing endogenous IFN-� on thedevelopment of ConA-induced hepatitis and also

tried to modulate the course of the disease by IFN-�treatment.

The results clearly demonstrated that developmentof ConA-induced hepatitis is abrogated by prophy-lactic treatment with IFN-� R-Ig fusion proteinand concordantly exacerbated by exogenously admin-istered IFN-�. Simultaneously, both reagents pro-foundly and differently modified, in both a quantitativeand qualitative fashion, the circulating cytokine profileof ConA-injected mice.

RESULTS

TABLE 1. Exacerbation of murine ConA-induced hepatitis by exogenously administered rat IFN-�

Groups (n) ConA (mg/kg) Treatment Frequency Tempo* ALT (U/l)

A (14) Nil PBS twice �24 h, +1 h 49�2B (20) 20 PBS (200 �l) twice �24 h, +1 h 4563�245C (20) 20 IFN-� (5�104) twice �24 h, +1 h 9321�567a

D (12) 20 IFN-� (105) twice �24 h, +1 h 13012�761a

E (18) 20 IFN-� (5�104) once �24 h 6913�1012F (14) 20 IFN-� (5�104) once +1 h 6123�931G (16) 20 IFN-� (5�104) once �1 h 5437�896H (18) 10 PBS (200 �l) twice �24 h, +1 h 345�41I (15) 10 IFN-� (5�104) twice �24 h, +1 h 6121�321b

J (16) 5 PBS (200 �l) twice �24 h, +1 h 71�15K (16) 5 IFN-� (5�104) twice �24 h, +1 h 103�28L (16) 5 IFN-� (105) twice �24 h, +1 h 5412�31c

M (12) 2.5 PBS twice �24 h, +1 h 67�16N (10) 2.5 IFN-� (105) twice �24 h, +1 h 165�56

*Relative to ConA-injection.For statistical analysis each group is compared to the relative PBS-treated control group.aP<0.0001 vs group B; bP<0.0001 vs group H; cP<0.0001 vs group K.The rate of mortality was 3/20 in group B, 6/20 in group C, 4/12 in group D, 4/18 in group E, 2/14 in group F, 3/16 in group G, 1/16in group I. No mice died in groups A, H, J–N.

Serological and histological signs of ConA-induced hepatic injury are exacerbated byexogenous IFN-�, that can also amplify thehepatitis inducing effects of subhepatitogenicdoses of ConA

As previously reported by ourselves andothers11,13,19 a substantial number of mice die within8 h after ConA-injection. In our own experience thislethal effect of ConA is not related to the extent of thehepatic lesions and occurs with significantly higherfrequency in females than in males, though we do notknow the reason for this biased sex distribution. In thepresent study there was a clear trend towards increasedmortality in the group of mice challenged with 20 mg/kg ConA and treated with IFN-� (see legend to Table1). Dead mice were not considered for serological andhistological analyses. As expected, acute signs of liverdamage were found at sacrifice in all the mice of thecontrol group injected with ConA and treated withPBS. These consisted of marked elevations of ALT inthe plasma and severe lobular infiltration with neutro-phil granulocytes, lymphocytes and monocytes, the

Essential pathogenetic role for interferon / 317

inflammatory process was also detected both in theportal areas and around the central veins (data notshown). Moreover, diffuse hepatocytic necrosis con-taining neutrophil granulocytes was also observed.Administering IFN-� 24 h prior to and 1 h after ConAinjection markedly exacerbated in a dose-dependentfashion these histological signs of hepatic damage thatcorrelated well with the significantly higher levels oftransaminases found in these mice as compared to thecontrols (data not shown and Table 1). Treating themice with IFN-� one single time at 24 h or 1 h prior toor 1 h after ConA-injection exacerbated to some extentthe hepatitis-inducing effects of ConA, but was lesseffective than the double treatment. The effect did notreach statistical significance, most likely because of thevariable individual response of the mice (data notshown and Table 1).

In another set of experiments mice were treatedwith lower doses of ConA (10 or 5 mg/kg) that wereknown from previous experiments to be capable ofonly inducing minimal, if any, serohistological signs ofhepatic damage, and evaluated whether pretreatmentwith IFN-� amplified the hepatitogenic potential ofthese low doses of ConA. As shown in Table 1, themice treated with 10, 5 or 2.5 mg/kg ConA onlyshowed slight dose-dependent increases in the bloodlevel of transaminases with minimal or no histologicalsigns of inflammatory infiltration of the livers (notshown). In contrast, when mice injected with 10 or5 mg/kg ConA were pretreated with IFN-� 24 h priorto and 1 h after the challenge, these mice developedhepatitis with serohistological parameters similar tothose induced by an optimal dose (20 mg/kg) of ConA(data not shown and Table 1). Note, however, that thehighest dose of IFN-� was necessary to provoke hepa-titis in mice receiving 5 mg/kg ConA. No effect wasnoticed when IFN-� pretreatment was given to micesubsequently injected with 5 mg/kg ConA. The neces-sity of a minimal dose of ConA under the threshold ofwhich IFN-� could not exert pro-inflammatory effectswas further demonstrated by the observation thatseven NMRI mice treated daily for two consecutive

weeks with 100 000 U IFN-�/day has ALT values andhistological appearance of the liver similar to those ofseven PBS-treated control mice (data not shown).

Prophylactic, but not therapeutic blockage ofendogenous IFN-� with either anti-IFN-� mAb orIFN-� R-Ig fusion protein abrogates developmentof ConA-induced hepatitis

In agreement with the exacerbating effects ofexogenously-administered IFN-� on the course ofConA-induced hepatitis, it was found that treatingthe mice prior to ConA-challenge either with AN-18anti-IFN-� mAb or the MoIFN-�R-M�2a offered asignificant protection against the biochemical and his-tological signs of hepatic injury (data not shown andTable 2). The protective effects of these IFN-� in-hibitors were lost when treatment was given afterConA-challenge, these mice showing serological andhistological signs of hepatic damage similar to those ofthe control animals (data not shown and Table 2).

TABLE 2. Prevention of ConA-induced hepatitis by blockage of endogenous IFN-� or IL-4

Groups (n) Treatment Frequency Tempo* ALT (U/l) Mortality

O (15) PBS once �1 h 6115�316 2/15 (13.3%)P (12) AN-18 mAb once �1 h 231�31a 0/12Q (12) IFN-� R-Ig once �1 h 156�19a 0/12R 10 AN-18 mAb once +2 h 5843�781 1/10 (10%)S 10 IFN-� R-Ig once +2 h 6975�913 0/10T 11 11B.11 mAb once �24 h 269�19a 0/11

Male NMRI mice were challenged i.p. with 20 mg/kg ConA. The mice were treated with AN-18 anti-IFN-� mAb (1 mg), theIFN-� R-Ig fusion protein MoIFN-�R- M�2a (300 �g) or 11B.11 anti-IL-4 mAb (0.75 mg) either before or after ConA challengeat the time points indicated in Table 2. For statistical analysis each group is compared to PBS-treated control mice. aP<0.0001 vsgroup O.

Blockage of endogenous IL-4 with mAb alsoprevents ConA-induced hepatitis

Because IFN-� and IL-4 most often display antag-onistic effects on the modulation of immuneresponses,24 the central pathogenetic role of IFN-� inthis model8–10, present paper), is in apparent contrastwith the resistance to the hepatitis inducing effects ofConA of mice with disruption of the IL-4 gene as wellas to the antihepatic effects of anti-IL-4 mAb.16 Toconfirm further the pro-inflammatory role of endogen-ous IL-4 in ConA-induced hepatitis, another set ofexperiments was performed where NMRI mice weretreated with anti-IL-4 mAb before challenge withConA. In agreement with the previous study,16 thistreatment markedly reduced the development of hepa-titis induced by ConA, both at the serological andhistological levels, as compared to the control group ofmice (data not shown and Table 2). EndogenousIFN-� and IL-4 are thus both key cytokines in thepathogenesis of ConA-induced hepatitis.

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Treatment with IFN-� or IFN-� R-Ig fusionprotein profoundly modulated blood cytokines inConA-challenged mice

Because ConA-induced hepatitis develops withsimultaneous elevation in the circulation of severalmacrophage and T-cell derived cytokines,11–19 andsince this may control or reflect the progression of theimmunoinflammatory syndrome, we next examined ifIFN-� or MoIFN-�R-M�2a interfered with the sys-temic levels of these molecules. Three groups of micetreated with PBS, IFN-� or MoIFN-�R-M�2a werekilled before ConA-injection and 2 and 8 h thereafter.As shown in Figure 1, IL-2, IL-4, IL-6, IL-10, IFN-�,and TNF-�, which were undetectable in the circulationof the mice at T0 (except for IFN-� that was measur-able in three out of seven mice treated with thiscytokine 24 h before), all appeared with different kin-etics in the blood of the PBS-ConA treated mice. Thiscirculating cytokine profile was profoundly modifiedby manipulation of endogenous IFN-�, in that themice pretreated with IFN-� showed significantly aug-mented circulating levels of IL-4, IL-6, TNF-� andIFN-�, and decreased levels of IL-10 (Fig. 1), whilethose receiving MoIFN-�R-M�2a showed augmentedblood levels of IL-10 and IFN-� and diminished levelsof IL-4, IL-6 and TNF-�. There were no significantdifferences in the plasma levels of IL-2 between theexperimental and control group of mice.

DISCUSSION

This study demonstrated that ConA-inducedhepatitis is prevented by blockage of endogenousIFN-� either with a mAb or with the MoIFN-�R-M�2a and concordantly exacerbated by exogenously-administered IFN-�. The disease promoting effects ofIFN-� in this model were further substantiated bythe possibility to fully induce serohistological signsof hepatitis when mice injected with otherwise sub-hepatitogenic doses (10, 5 mg/kg) of ConA were pre-treated with IFN-�. These data confirm, complementand extend previous reports on the essential patho-genetic role of endogenous IFN-� in ConA-inducedhepatitis8–10 and offer some mechanistic insights intothe mode of action of IFN-� and its specific inhibitorsin the exacerbation/prevention of this immunoinflam-matory condition.

It is well known that at least two major subsets ofcytokines exist in mice, rats and humans that areproduced by several lymphoid and nonlymphoid cellssuch as T and B cells, macrophages, NK cells, endo-thelial cells, epithelial cells and mastocytes.25 Type 1cytokines such as IL-1, IL-2, IL-12, IL-18, TNF-�/�and IFN-� stimulate cytotoxic T cell and macrophage

functions, favour Ig isotype and subclass switching toIgG2a and IgG3, are involved in DTH-responses, andare thought to play a major role in the pathogenesis oforganic specific autoimmune diseases such as type 1diabetes, multiple sclerosis, and autoimmune thyroidi-tis.1,25 On the contrary, the type 2 cytokines IL-4, IL-5,IL-6, IL-10 and IL-13 mostly exert a downregulatoryeffect on cytotoxic T cell and macrophage responses,stimulate IgG1 and IgE production and are involved inallergic reactions and, perhaps, autoantibody-mediatedsystemic autoimmune diseases such as rheumatoidarthritis and systemic lupus erythematosus (SLE).25

Since an opposite and often antagonistic functionexists between type 1 and type 2 cytokines, it has beenproposed that the natural course, and perhaps even theappearance of organ-specific immunoinflammatorydiseases may be favoured by or depend on an exagger-ated production of type 1 cytokines opposed or sec-ondary to a deficit in type 2 cytokine production (see26

for a review).However, recent evidence suggests that this

hypothesis may be oversimplistic to account for thecomplex events responsible for autoimmunity, and ledto revisiting the type 1/type 2 cytokine paradigm in thissetting.27 This work with ConA-induced hepatitis sup-ports the limit of the above pathogenetic concept. Inthis immunoinflammatory disease model, the contem-poraneous pathogenetic contribution of the two proto-typical type 1 and type 2 cytokines IFN-� and IL-4 isproved by the equally preventive effects of blockageendogenous IFN-� or IL-4 with specific inhibitors suchas neutralizing mAbs or an IFN-� R-Ig fusion protein.That, in the present study, the exacerbating effects ofexogenous IFN-� were associated with markedlyincreased blood levels of IL-4, and that the antihepaticeffects of MoIFN-� R-Ig fusion protein were con-versely accompanied by reduction in circulating IL-4further suggest that the pathogenetic action of IFN-�may be at least partly mediated by IL-4. Although thecellular source of augmented IL-4 production inducedby IFN-� treatment is not known, likely candidates areliver NK1+ T cells that are involved in ConA-inducedhepatitis through production of TNF-� and IL-4,16 thesecretion of the latter from these cells being favouredby IL-12, a cytokine that is induced by IFN-�.28 To thebest of our knowledge these are the first data showingin vivo synergism between IFN-� and IL-4 in inductionof organ-specific autoimmune disease. These data fit inwith our previous study in an experimentally-inducedmodel of SLE where the disease promoting effects ofIFN-� were associated with increased blood levels ofIL-4 and augmented numbers of type 2 cytokine (IL-4,IL-6) secreting lymphoid cells.29 How IFN-� and IL-4interact in promoting ConA-induced hepatitis has notbeen ascertained. Likely candidates are some immuno-stimulatory properties shared by the two cytokines

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Figure 1. Modulation by IFN-� and IFN-� R-Ig fusion protein of ConA-induced cytokine release.

Seven mice from each group were sacrificed prior to (T0) and 2, and 8 h after injection of ConA, and plasma samples from individual mice wereused for cytokine measurements. IFN-�, (50 000 U/mouse) was administered 24 h prior to and 1 h after ConA-challenge, MoIFN-�R-M�2a andPBS 1 h prior to ConA-challenge. Data are shown as means�SEM. For statistical analysis each group is compared to PBS-treated control mice.a, P=0.018; b, P=0.044; c, P=0.02; d, P=0.04; e, P<0.0001; f, P<0.0001; g, P=0.004; h, P<0.0001; i, P<0.0001; j, P=0.0021; k, P=0.02 byANOVA. (——) PBS; (– – –) IFN-�; (- - - -) IFN-� fusion protein.

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such as induction of MHC class II antigen expressionand some integrins on macrophages as well as VCAMon endothelial cells.24. Moreover, both cytokines canaugment the production of TNF-�, that activatescaspase-1-induced apoptosis of hepatocytes.18

The impact of IFN-� and MoIFN-� R-Ig fusionprotein on the circulating cytokine profile of ConA-injected mice largely accorded with known immuno-biological properties of this cytokine1,2 and fit in withthe pro- and anti-inflammatory effects respectivelyobserved. Relative to ConA-challenged control mice,the increase in the blood levels of TNF-� and thedecrease in those of IL-10 in IFN-� treated mice wasparticularly striking. These findings were not un-expected as IFN-� is known to up-regulated TNF-�production and decrease that of IL-10 under differentexperimental conditions.1,2,30 Since TNF-� and IL-10exert pro- and anti-inflammatory effects on the devel-opment of ConA-induced hepatitis12,14,17,18 it seemslikely that the impact of IFN-� or MoIFN-� R-Igfusion protein on the outcome of this disease may berelated to the changes they induced in the blood levelsof these two cytokines.

The slight, though significant increase in the bloodlevels of IL-6 observed in ConA-injected mice treatedwith IFN-� as compared to the control mice does notapparently fit in with the protective effects of exogen-ous IL-6 in this model.12 Perhaps the capacity of IFN-�to augment IL-6 blood levels in these mice limitedits pathogenetic effects, without being sufficient tocounteract them completely because of the contem-poraneous pro-inflammatory effects it induced. Forexample, the antihepatic effects of IL-6 may have beenantagonized by the excessive release of TNF-� in theblood and/or could have required greater circulatinglevels of IL-10 to be present for some degree ofprotection to be exerted. In the same manner, the lowerblood levels of circulating IL-6 observed in MoIFN-�R-M�2a-treated mice as compared to the controlsmay be diminishing its protective effects. Note thatthese data are in apparent conflict with our previousstudy showing augmented IL-6 production fromsplenic lymphoid cells of anti-IFN-�-treated NODmice.4 The different experimental conditions (in vivo vsex vivo) used and the different animal models mayprobably explain these discrepant results.

No significant differences were observed in theblood levels of IL-2 between the three groups of micechallenged with ConA and treated with PBS, IFN-�and MoIFN-�R-M�2a. This accords with the lack ofeffect of IFN-� on IL-2 gene modulation either at thetranscriptional or mRNA level.1 If and how this lackof effect has influenced the capacities of the tworeagents to modulate the course of ConA-inducedhepatitis requires more information on the role, if any,of endogenous IL-2 in this model.

Since the AN-18 mAb used in the solid-phaseELISA to measure mouse IFN-� does not bind, or onlypoorly binds rat IFN-�31 the amount of circulatingIFN-� quantitated by ELISA only reflects the endog-enous cytokine of the mouse and not the exogenously-administered rat IFN-�. The marked increase in theblood levels of IFN-� observed in ConA-challengedmice treated with the cytokine as compared to thecontrol animals bespeaks an augmented synthesis/release of endogenous IFN-� induced in an autocrinefashion by treatment with rat IFN-�. This enhance-ment may have occurred via IL-12, a heterodimericcytokine that augments the production of IFN-� thatin turn stimulates IL-12 production.28 IL-12 appears inthe blood during ConA-induced hepatitis17 and studiesare in progress to ascertain the interrelation betweenIFN-� and IL-12 on the course of this disease model.Relative to the control mice a significant increase in thecirculating levels of IFN-� could also be noticed inConA-challenged mice treated with MoIFN-�R-Igfusion protein. That IFN-� was measurable in thecirculation of these mice was not surprising becauseMoIFN-�R-M�2a binds different epitopes than theAN-18 and XMG 1.2 mAbs used for the ELISA.Accordingly, IFN-� was below the limit of sensitivityof the assay in ConA-injected mice treated with theAN-18 mAb (data not shown). The augmented bloodlevels of IFN-� in MoIFN-�R-M�2a-treated miceprobably mirrors its augmented production that mightbe aimed at counteracting the functional deficit inIFN-�, as binding to the surface expressed, biologicallyactive receptor that is required for signal transductionis inhibited by the MoIFN-�R-M�2a injected.

Although our data demonstrated that treatmentwith IFN-� polarises the endogenous circulatingcytokine in ConA-injected mice towards a diseasepromoting phenotype with increased levels of patho-genetic IL-416 and TNF-�12,16,18 and diminishedamounts of the protective IL-10, they do not dismantlethe precise cellular and molecular mechanisms bywhich this cytokine exerts its hepatotoxic effects. Thepleiotropic biological properties of IFN-� anticipate itsintervention at multiple steps throughout the pathwaysleading to hepatic damage in response to ConA.1,2

IFN-� may favour homing of mononuclear cells andneutrophils into the liver parenchyma by up-regulatingleukocyte-endothelial cell interactions via control ofexpression of adhesion molecules, chemokines andtheir receptors. It may promote the immunoinflam-matory damage of the hepatocyte in situ by up-regulating expressions of MHC class II and I antigensthat play a central role in (auto)antigen procession,presentation and destruction, respectively. In addition,Kusters et al., have demonstrated that IFN-� andTNF-� act in concert in inducing hepatic DNA frag-mentation and liver cell death, each cytokine being

low doses of streptozotocin at doses of 30 �g/mouse.

necessary.

Essential pathogenetic role for interferon / 321

necessary but not sufficient to provoke liver injury inConA-treated mice.8 Tagawa et al. have also suggestedthat IFN-� plays a pivotal role in ConA-inducedhepatitis by activating Fas-induced apoptosis of hepa-tocytes,10 but further studies have questioned therelevance of FasL/CD95-dependent processes in thedevelopment of ConA-induced hepatitis.18,32

The favourable effects of specific blockage ofendogenous IFN-� with mAbs on the course of severalimmunoinflammatory diseases in animal models haveprovided valuable rationale for novel therapeuticavenues in the clinical setting that selectively target theaction/production of IFN-�. As we discussed else-where,4 treating human patients with mAbs may bedifficult as these Abs are often of heterologous originand can thus provoke the formation of xenogenic Abscapable of reducing their biological effects.20,21 More-over, it has also been shown that fully humanized mAbmay still retain immunogenic properties.22 Recently,Garotta’s group have characterized and produced asoluble form of murine (sm)IFN-� R33 that is notimmunogenic in mice and is as active as anti-IFN-�mAbs in murine models of SLE.34 Our recent study inthe diabetes-prone NOD mouse has also confirmed thelack of immunogenicity of smIFN-� R and demon-strated it to possess an efficiency comparable to that ofAN-18 and XMG 1.2 anti-IFN-� mAbs in preventingspontaneous autoimmune diabetes in NOD mice.4

However, we also observed that, unlike the anti-IFN-�mAbs, the smIFN-� R was ineffective in the acceler-ated model of diabetes that can be induced in NODmice by one large dose of CY. Because CY-induceddiabetes seems to be more aggressive than that spon-taneously occurring in NOD mice, we interpreted thelimited efficacy of smIFN-� R in this setting as aconsequence of its shorter half-life (2–4 h after i.p.injection) than the anti-IFN-� mAbs (approximately 7days), and suggested that IFN-� R-Ig fusion proteinsshould be more suitable for in vivo treatment. Theavailability of the IFN-� R-Ig fusion protein MoIFN-�R-M�2a that is also devoid of immunogenic proper-ties and possesses a longer half life of the smIFN-� Rhas therefore allowed us to test its effects in murineConA-induced immunoinflammatory hepatitis. As inmice with multiple low dose streptozotocin-induceddiabetes,35 this reagent also exerted very powerfulprotective effects against serohistological signs ofConA-induced hepatitis and in a manner very similarto those achieved by AN-18 anti-IFN-� mAb. Thesedata provide further in vivo evidence for the potentialuse of IFN-� R/fusion protein as an alternativemeans for antagonizing this cytokine in human IFN-�dependent immunoinflammatory diseases such asautoimmune hepatitis.

MATERIAL AND METHODS

Rat IFN-�Rat IFN-� (IFN-)� was produced by a CHO cell line

transformed with a vector expressing the chromosomal geneencoding rat IFN-� as described elsewhere.36 Endotoxincontamination was less than 80 pg/ml/106 U as determined inthe Limulus amebocyte lysate assay. IFN-� was diluted inPBS and injected intraperitoneally (i.p.) into mice at a dose of5�104 U/mouse. This or similar doses of mouse/rat IFN-�have been previously found by ourselves and others to exertbiological effects in murine models of rheumatoid arthritis37

and systemic lupus erythematosus.29,38

AN-18 rat monoclonal Ab 1gG2a anti-mouseIFN-�

This monoclonal (m)Ab, characterized by Prat et al.31

was produced as described elsewhere.3 AN-18 neutralizes theactivity of both natural and recombinant mouse IFN-�, butdoes not react with murine IFN-� or murine IFN-�. Theneutralizing titre of the AN-18 mAb is 1�105 units mouseIFN-� mg/proteins as assessed by its ability to suppress thecapacity of IFN-� to reduce the cytopathic effect producedby encephalomyelitis virus on L929 cells. We have previouslyshown that this mAb modulates the course of experimentalautoimmune thyroiditis,3 type II collagen induced arthritis,37

spontaneous and Cyclophosphamide (CY)-induced diabetesin NOD mice,4 and experimental autoimmune orchitis.6 Anisotype-matched control for the AN-18 mAb has not beenused as the biological activity of this mAb is well proved andsuch control is no longer considered necessary.

Mouse IFN-� R-Ig fusion proteinMouse IFN-� R-Ig fusion protein consisting of mouse

�2a chain constant domains fused to the carboxyl terminus ofthe soluble mouse IFN-� R (MoIFN-�R-M�2a) was con-structed as described elsewhere.38 MoIFN-�R-M�2a has ahalf-life of 41–42 h blood persistency after i.v. or i.p. injec-tion, respectively, does not induce Ab formation in mice andprevents immunoinflammatory diabetes induced by multiple

35

11B.11 rat mAb IgG1 anti-mouse IL-4 mAbHybridoma cells producing rat IgG1 anti-mouse IL-4

Ab (11B.11) were obtained from ATCC. Hybridoma cells(3�106) were injected into nude BALB/c mice, ascites werecollected after 3 weeks and immunoglobulins isolated using aprotein G column (Pharmacia Biotech, Milan, Italy). ThemAb was diluted in PBS and 0.75 mg injected i.p. into micein a 200 �l volume. This dose of 11B.11 mAb, which neutral-ized 1300 U of mIL-4 in the CT.4S bioassay, has beenpreviously demonstrated to exert biological effects in murinemodels of arthritis.40–42 As for the AN-18 mAb, an isotype-matched control for the 11B.11 mAb is no longer considered

42

Mice and hepatitis inductionSix to eight weeks old male Naval Medical Research

Institute (NMRI) albino mice were purchased from CharlesRiver (Calco, Italy). The mice were kept under standard

322 / Nicoletti et al. CYTOKINE, Vol. 12, No. 4 (April, 2000: 315–323)

laboratory conditions (non-specific pathogen free) at 24�Cwith free access to food and water. The food was withdrawn16 h prior to the experiments. ConA (Sigma Chemical,St Louis, MO, USA) was dissolved in sterile phosphatebuffered saline (PBS) and injected to mice via the tail vein.The groups were treated i.p. with PBS, IFN-�, AN-18anti-IFN-� mAb, MoIFN-�R-M�2a or 11.B11 anti-IL-4mAb according to the experimental design shown in Table 1.An additional control group consisted of mice challengedonly with PBS (Table 1). Since marked increases of trans-aminase activities along with severe histological signs ofhepatic injuries have been reported to develop 8 h after ConAinjections in these mice,1,2 the animals were sacrificed after8 h, and blood and livers were collected.

Assay for plasma transaminase activitiesPlasma alanine aminotransferase (ALT) activity was

determined by a standard photometric assay using a bio-chromatic analyser.

Cytokine measurementsPlasma samples from individual mice were collected for

cytokine measurement. IL-2, IL-4, IL-6, IL-10, and TNF-�were quantitated by solid phase ELISA kits for detection ofmouse cytokines purchased from Genzyme (Cambridge, MA,USA). Samples were run in duplicates according to themanufacturer’s instructions. IFN-� was also quantitated bystandard solid-phase ELISA developed by using anti-IFN-�Ab pairs consisting of purified AN-18 mAb and biotynilatedXMG 1.2 mAb Pharmingen (San Diego, CA, USA). Thelower limit of sensitivity of the assays were 2 pg/ml forIFN-�, 5 pg/ml for IL-4, 10 pg/ml for IL-10, 12 pg/ml forIL-6, and 15 pg/ml for IL-2 and TNF-�. To calculate meanvalues, samples with cytokine values below the level ofdetection were assigned the limit of sensitivity of the assay astheoretical values.

Histological examinationEight hours after ConA injection, the livers were

removed, fixed in 10% formalin, embedded in paraffin, slicedin 5 �m sections and stained with haematoxylin and eosin(HE) for histological examinations at 125-fold magnification.This was performed by an observer unaware of the treatmentof the mice.

Calculation of dataResults are expressed as mean values�SEM. Statistical

analysis was performed by ANOVA.

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