Dichotomic effects of IFN-γ on the development of systemic lupus erythematosus-like syndrome in MRL-lpr / lpr mice

0014-2980/00/0202-438$17.50+.50/0 © WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2000

Dichotomic effects of IFN- q on the development ofsystemic lupus erythematosus-like syndrome inMRL-lpr/lpr mice

Ferdinando Nicoletti1, Roberto Di Marco2, Paola Zaccone2, Ming Xiang2, GaetanoMagro3, Sebastiano Grasso3, Stefania Morrone4, Angela Santoni4, Yehuda Shoenfeld5,Gianni Garotta6 and PierLuigi Meroni7

1 Institute of Microbiology, University of Milan, Milan, Italy2 Institute of Microbiology, University of Catania, Catania, Italy3 Institute of Anatomopathology, University of Catania, Catania, Italy4 Department of Experimental Medicine, University La Sapienza, Rome, Italy5 Research Unit of Autoimmune Diseases, Department of Medicine B, Sheba Medical Center,

Tel-Hashomer, Israel6 Ares Serono, Geneve, Switzerland7 Allergy and Clinical Immunology Unit, IRCCS, Istituto Auxologico Italiano, Milan, Italy

Systemic lupus erythematosus (SLE)-prone female MRL-lpr/lpr (MRL-lpr) mice were treatedwith mouse or rat IFN- + under different experimental conditions, both prophylactically in 6-to 8-week-old animals and therapeutically in 12- to 18-week-old SLE-affected mice. It wasfound that IFN- + heterogeneously modulated the course of the disease in MRL-lpr mice.When administered prophylactically, IFN- + favorably modulated the histological, serologicaland clinical signs of the disease. Relative to untreated or PBS-treated control animals, theMRL-lpr mice which received IFN- + were virtually free of inflammatory infiltration of the kid-neys and the lungs, had lower levels of azotemia with reduction of both circulating IgG1,IgG2a and IgG3 and anti-double strand (ds) and single strand (ss) DNA antibodies, milderskin vasculitis, significantly reduced enlargment of their lymph nodes and lower weight of thespleens. IFN- + also lowered the rate of mortality of MRL-lpr mice. In contrast to these find-ings, therapeutically administered IFN- + worsened the course of the disease in MRL-lprmice, which exhibited increased proteinuria, higher levels of IgG2a and IgG3 and anti-ds and-ss DNA antibodies, more aggressive nephritis and died at an earlier age than PBS-treatedcontrol mice. The dichotomic effect of IFN- + on disease manifestation in MRL-lpr mice offersnew insights into the complex role of this cytokine in the regulation of systemic autoimmunitysuch as SLE.

Key words: Autoimmune diseases / Cytokine / IFN- + / MRL-lpr mouse / Systemic lupus erythema-tosus

Received 16/3/99Revised 30/9/99Accepted 19/10/99

[I 19407]

Abbreviations: CIA: Collagen-induced arthritis EAN:Experimental autoimmune neuritis EAT: Experimental auto-immune thyroiditis MRL-lpr: MRL-lpr/lpr IDDM: Insulin-dependent diabetes mellitus ds: Double stranded ss: Sin-gle stranded SLC: Splenic lymphoid cells NOD: Nonobesediabetic KO: Knockout

1 Introduction

MRL/lpr-pr (MRL-lpr) mice spontaneously develop asystemic, CD4+ T cell/macrophage-dependent autoim-mune disease with histopathological and clinical fea-tures similar to human SLE, including B lymphocyte

hyperactivity with hypergammaglobulinemia, autoanti-body production and deposition of immune complexesin vital organs leading to fatal glomerulonephritis andvasculitis (see [1] for reviews). Moreover, these mice alsoexhibit massive proliferation of atypical CD3+ B220+ andCD3+ CD4–, CD8– T cells caused by a defect in the Fasantigen which mediates apoptosis [1].

Since cytokines are important mediators of T cell andmacrophage function, their abnormal production mightbe involved in the SLE-like syndrome of MRL-lpr mice.Two major subsets of cytokines exist, namely type 1 andtype 2, which possess different and often antagonisticfunctions. Type 1 cytokines (IL-2, IL-12, TNF- § , IFN- + )

438 F. Nicoletti et al. Eur. J. Immunol. 2000. 30: 438–447

Table 1. Effects of IFN- + prophylaxis on the clinical signs of 17-week-old MRL-lpr micea)

Treatment n Body weight (g) Skin lesions (%)b) Lymph node swelling Proteinuria

IFN- + 20 35.8 ± 2.9* 0.3 ± 0.1* (20 %) 1 ± 0.4* 1.1 ± 0.3*

PBS 20 40.6 ± 1.8 3.1 ± 0.6 (85 %) 2.9 ± 1.1 2.8 ± 0.7

Nil 20 41.3 ± 1.9 2.6 ± 0.6 (85 %) 2.9 ± 0.9 3 ± 1.2

a) Results are shown as mean values ± SD.b) Incidence of skin lesions.*, p X 0.0001 vs. PBS-treated mice by Mann-Whitney U test.

stimulate macrophage and cytotoxic cells, favor IgG2aand IgG3 production and are implicated in thepathogenesis of organ-specific autoimmune diseasessuch as type 1 diabetes (see [2, 3] for reviews). Type 2cytokines (IL-4, IL-5, IL-6, IL-10, IL-13) stimulate Blymphocyte functions, induce the production of IgG1and IgE, promote allergic reactions and arepathogenetically involved in autoantibody-mediated,systemic, autoimmune diseases such as rheumatoidarthritis and SLE [2, 3]. However, conflicting results havebeen obtained as to the altered type 1/type 2 cytokinebalance in SLE. In human SLE, the augmentedproduction of IL-6 and IL-10 and the diminishedproduction of IL-2, IL-12, TNF- § and IFN- + [2, 4]suggests a role for type 2 cytokines in the developmentof the disease, and so does the central role of IL-4 in alupus-like syndrome in murine graft-versus-hostand host-versus graft reactions [2]. The type 1/type 2cytokine hypothesis is also controversial in(NZBxNZW)F1 mice, where the disease can equally beprevented by anti-IFN- + [5] or anti-IL-10 [2] mAb, and inMRL-lpr mice. In this latter model we and others havefound that the production of IL-4 and IL-6 is increasedand that of IL-2 and IFN- + is reduced as compared toother strains [2, 6]. Moreover, blockade of endogenousIFN- + with specific mAb improves neither clinical norhistological signs of the disease [7, 8], the developmentof which is suppressed by infection with an IL-2/vacciniarecombinant virus [2]. In contrast, others found thatMRL-lpr mice produce more IFN- + than IL-4 and IL-10during disease development [9], and that exogenouslyadministered IL-12 (a powerful IFN- + inducer [3])accelerates glomerulonephritis development in theseanimals [10]. Along with the ability of IFN- + to induce theproduction of nephritogenic IgG3 [11], these latterfindings all incriminate IFN- + and, by extension, type 1cytokine-secreting cells as important effectors of theautoimmune diathesis of MRL-lpr mice. We have alsorecently found that exogenous IFN- + exacerbated thecourse of the disease in an induced model of murine SLE[12].

However, that MRL-lpr mice with a disrupted IL-4 orIFN- + gene [IL-4 knockout (KO), IFN- + KO] [13–14] orthat do not express the IFN- + R [15–16] develop milderserohistological and clinical signs of disease as com-pared to “normal” MRL-lpr mice indicates that thesecytokines are both essential for development of lupus-like disease in these animals. This also suggests that the“type 1/type 2 cytokine” hypothesis may be too over-simplified to explain the complex events leading todevelopment of SLE-like syndrome in MRL-lpr and thatboth subsets may cooperate to the autoimmune diathe-sis of these mice.

Here we examined the effects of prolonged administra-tion of murine/rat IFN- + in MRL-lpr mice at differenttimes. IFN- + exerted dichotomic actions on diseasedevelopment, the early prophylactic treatment exhibitingfavorable effects and the late institution of treatmentaccelerating disease development.

2 Results

2.1 Early prophylactic treatment with IFN- qreduces serological and histoimmunologicalsigns of disease and reduces the rate ofmortality in MRL-lpr mice

In a first set of experiments, 6-week-old female MRL-lprmice were treated with murine IFN- + , at the dose of80 000 U/mouse/day six times a week for 11 consecu-tive weeks. Control, sex- and age-matched MRL-lprmice were either untreated or treated under the sameexperimental conditions with PBS. At the age of 17weeks the treatment was discontinued. Twenty micefrom each group were examined for the clinical indices ofdisease shown in Table 1. All the mice from each groupwere followed thereafter for mortality until the age of 32weeks. Another three groups of MRL-lpr mice, treatedas above, were killed at 17 weeks for serological andhistoimmunological analyses (Table 2).

Eur. J. Immunol. 2000. 30: 438–447 Dichotomic effects of IFN- q on the development of systemic lupus 439

Fig. 1. Effects of IFN- + prophylaxis on SLC subsets in MRL-lpr mice. SLC from 17-week-old female MRL-lpr micetreated six times a week for 11 consecutive weeks witheither 80 000 U mouse IFN- + ( ß ; n = 4) or PBS ( | ; n = 5)were incubated with different FITC- or PE-conjugated ratmAb recognizing mouse T (Thy-1.2), Ig+ and NK cells andthe L3T4+ (CD4+), Ly-2 (CD8), B220+, CD45+ Ia+ and CD25+

subsets. Results are expressed as % ± SD of cells.

Table 2. Effects of IFN- + prophylaxis on murine lupus in 17-week-old MRL-lpr micea)

IFN- + (n = 16) PBS (n = 18) Nil (n = 12)

Anti-ds DNA Ab (A)b) 0.3 ± 0.3* 1.4 ± 1 1.2 ± 0.5

Anti-ss DNA Ab (A)b) 0.7 ± 0.6* 2 ± 1.1 2.2 ± 1

Spleen weight (g) 0.3 ± 0.2** 0.5 ± 0.2 0.5 ± 0.3

Cells/spleen (× 106) 213 ± 99* 512 ± 101 498 ± 111

Azotemia 20 ± 5* 37 ± 6 38 ± 8

Nephritic lesion scores 0.3 ± 0.3* 3.1 ± 1 3.7 ± 0.9

Pulmonary lesion scores 6.8 ± 4* 30.1 ± 10 28.9 ± 8

a) Results are shown as mean values ± SD.b) 1/1000 serum dilution.*, p X 0.0001; **, p X 0.004 vs. untreated mice by Mann-Whitney U test.

As expected, clinical, histological and biochemical signsof SLE-like syndrome were observed in control micewhich all exhibited severe proteinuria, diffuse skinlesions, swelling of lymph nodes and abnormally ele-vated body weight (Table 1); simultaneously, histoimmu-nological and serological analyses revealed massivemononuclear cell infiltration of the kidneys and lungs,elevated levels of azotemia, splenomegaly, hypergam-maglobulinemia, increased titers of anti-double-stranded (ds) and anti-single-stranded (ss) DNA andaugmented frequency of splenic CD3+B220+ T cells(Tables 1 and 2 and Fig. 1). In contrast, the mice treatedwith IFN- + exhibited reduced swelling of lymph nodes,milder skin lesions and lower body weight (Table 1).Moreover, histological and serological studies demon-strated that IFN- + -treated MRL-lpr mice had significantlylower nephritic and pulmonary lesion scores, diminishedazotemia, lower serum titers of anti-ds and -ss DNA Ab,decreased circulating levels of total IgG, IgG1, IgG2aand IgG3, and lower weight of the spleens with reducednumbers of lymphoid cells than control animals(Tables 2, 3). However, no differences could be foundbetween IFN- + - and PBS-treated control mice usingimmunofluorescence analyses, though the mice from theformer group exhibited a slight, not significant, reductionin the number of atypical CD3+B220+ T cells, as well asof T and B cells along with an increase of Ia+ spleniclymphoid cells (SLC) and NK cells as compared toPBS-treated control mice (Fig. 1).

Finally, the life-span of IFN- + -treated mice was signifi-cantly longer than that of the mice from the two controlgroups which were either treated with PBS or were leftuntreated (Fig. 2). However, the preventive effect of IFN-+ was only temporary and a rate of mortality similar to

that of control mice was reached by the age of 32 weeks,e.g. 15 weeks after IFN- + was discontinued (Fig. 2).

Since IFN- + has pleiotropic effects, some of which areconcentration- or dose dependent, another experimentwas conducted where 8-week-old MRL-lpr mice weretreated with a different dose of either rat IFN- + (100 000U/mouse) or PBS administered under a slightly modifiedearly prophylactic regimen, i.e. three times weekly onalternate days until the age of 19 weeks. Since calorierestriction extends the life-span of MRL-lpr mice [17], itwas also important to rule out the possibility that the


Fig. 2. IFN- + prophylaxis increases the life-span of MRL-lprmice. Six-week-old female MRL-lpr mice were treated sixtimes a week for 11 consecutive weeks with 80 000 U/mouse murine rIFN- + (n = 35). Control sex- and age-matched MRL-lpr mice were either untreated (n = 35) ortreated under the same experimental conditions with PBS(n = 35). At the age of 17 weeks the treatment was discontin-ued and the mice from each group were followed thereafterfor mortality rate until the age of 32 weeks. p = 0.035 vs.untreated and PBS-treated mice by Logrank (Mantel-Cox).The data are merged from three independent experiments.Variability between each experiment was lower than 10 %.

Table 3. Effects of IFN- + treatment on circulating IgG1, IgG2a and IgG3 in MRL-lpr micea)

Groups (n) Treatment Weeks of age IgG1 IgG2a IgG3

A (16) IFN- + 6–17 3.1±0.9* 19.6± 8.9* 1.6±0.8*

B (18) PBS 6–17 9.2±3.9 40±20 4.3 ± 2.4

C (12) IFN- + 18–25 3.2±1.7** 45.9±17.5*** 6.6±1.4**

D (31) PBS 18–25 7.4±3.7 32.6± 9.4 3.8 ± 2.4

a) Serum samples were obtained from individual 17- or 25-weeks-old female MRL-lpr mice treated with either IFN- + (80 000 U/mouse six times a week) or PBS during the weeks of age described above. Results are expressed as mg/ml (mean values ±SD). *, p X 0.0001; **, p = 0.002; ***, p = 0.02 vs. PBS-treated mice by Mann-Whitney U test.

lower body weight observed in IFN- + -treated mice ascompared to the controls in the previous experiment wasdue to IFN- + toxicity, e.g. IFN- + -induced anorexia.Hence, 8-week-old “normal”, non-spontaneouslyautoimmune-prone CBA/J mice and euglycemicdiabetes-prone nonobese diabetic (NOD) mice weretreated three times weekly on alternate days until the ageof 19 weeks with either 100 000 U rat IFN- + or PBS.

Once again IFN- + exerted clear-cut beneficial effects onthe rate of survival of MRL-lpr mice. Accordingly, 60 %

(6/10) of the PBS-treated control mice died within the19th week of age (mean age of death ± SD = 128 ± 11.5days) as compared to none of the ten mice receivingIFN- + [p = 0.041 vs. PBS-treated mice by Logrank(Mantel-Cox)]. A lower dose of rat IFN- + (50 000 U)administered exactly as above was also capable ofreducing the rate of mortality at 19 weeks of age to 11 %(1/9, data not shown). Also in this experiment IFN- + -treated mice showed much better general appearancethan PBS-treated controls, with diminished enlargementof their lymph nodes and reduced, sometime absent,skin vasculitis (data not shown). Moreover, as in the firstexperiment, IFN- + -treated mice had significantly lowerbody weight than the control mice at different time pointsduring the treatment period (not shown). This wasunlikely to be due to a toxic effect of IFN- + since neitherCBA/J mice nor NOD mice treated under similar experi-mental conditions with rat IFN- + showed significant dif-ferences in body weights as compared to PBS-treatedmice (not shown). This concurs with our study in malediabetes-prone NOD mice treated with 80 000 U ratIFN- + given i.p. three times weekly from the 8th until the16th weeks of age, which exhibited body weights com-parable to that of the control mice throughout the studyperiod (unpublished observation and [18]).

2.2 Therapeutic treatment with IFN- q aggravatesglomerulonephritis and accelerates mortalityin MRL-lpr mice

To evaluate whether IFN- + applied therapeutically toSLE-affected animals could still have beneficial effects,18-week-old MRL-lpr mice, which had already devel-oped signs of the systemic autoimmune disease (seeTable 4), were treated daily six times a week for 7 con-secutive weeks with either PBS or mouse IFN- + (80 000U/mouse). Unexpectedly, under these conditions IFN- +accelerated both the tempo and cumulative rate of mor-tality (Fig. 3). Serological and/or histological analyseswere also performed in some mice before commenc-


Fig. 3. IFN- + therapy increases the mortality rate of MRL-lprmice. Eighteen-week-old MRL-lpr mice were treated daily,six times a week for 7 consecutive weeks with either PBS(n = 74) or 80 000 U IFN- + (n = 69). p X 0.0001 vs. PBS-treated mice by Logrank (Mantel-Cox). The data are mergedfrom four independent experiments. Variability betweeneach experiment was lower than 10 %.

Table 4. Clinical and serological parameters of 4- and 18-week-old MRL-lpr micea)

MRL-lpr mice 4 week oldn = 10

18 week-oldn = 10

Anti-ds DNA Ab (A)b) ND 1.5±0.4

Anti-ss DNA Ab (A)b) ND 2.5±0.6

Azotemia 16±7 36±9

Skin lesions (%)d) 0 (0 %) 2.4±0.5 (80 %)

Proteinuria 0 2.6±0.7

a) Results are expressed as mean values ± SD.b) 1/1000 serum dilution.c) Incidence of skin lesions.

ing the treatment at the age of 18 weeks, during thestudy at the age of 21 weeks and in all the survivors atthe end of the experiment at the age of 25 weeks(Table 5). While there were no significant differences inserological and/or immunological parameters in themice prior to the treatment, both at 21 and 25 weeks themice treated with IFN- + exhibited higher levels of azote-mia, more severe proteinuria and higher serum titers of

Table 5. Effects of IFN- + therapy on murine lupus in MRL-lpr micea)

Weeks of age 18 21 25

Treatment (number ofanimals)

IFN- + (25) PBS (30) IFN- + (25) PBS (25) IFN- + (12) PBS (31)

Anti-ds DNA Ab (A)b) 1.3±0.5 1.4±0.9 1.8±0.6* 1.2±0.6 2.3±1** 1.3±0.7

Anti-ss DNA Ab (A)b) 1.9±0.7 1.8±0.6 3.0±0.7*** 2.0±0.9 3.2±0.7** 2.1±0.6

Spleen weight (g) ND ND ND ND 0.5±0.1 0.4±0.3

Cells/spleen (× 106) ND ND ND ND 461±101 459±130

Azotemia 37±8 36±9 57±7*** 41±7 61±10*** 50±8

Proteinuria 2.4±0.9 2.5±0.8 3.7±0.6**** 3.0±0.7 3.6±0.7** 3.0±0.6

Nephritic lesion scores ND ND ND ND 4*** 3.0±0.8

Pulmonary lesionscores

ND ND ND ND 30.3±11 31.0±10

Skin lesions (%)c) 2.4±0.9 2.5±0.6 2.6±0.8 2.7±0.9 2.2±0.7**** 3.2±0.7

(76 %) (73.3 %) (100 %) (100 %) (66.7 %) (100 %)

a) Results are shown as mean values ± SD. For statistical analysis each group is compared to PBS-treated controls.b) 1/1000 serum dilution.c) Incidence of skin lesions.*, p X 0.013; **, p X 0.002; ***, p X 0.0001; ****, p = 0.001 by Mann-Whitney U test.

anti-ds and -ss DNA autoantibodies than PBS-treatedcontrols (Table 5). In addition, at 25 weeks of age, IFN- + -treated mice also exhibited a more severe mononuclearcell infiltration of the kidneys, but not of the lungs, than


PBS-treated controls (Table 5), and increased blood lev-els of total IgG2a and IgG3 with simultaneous reductionin IgG1 (Table 3). However, lymph node swelling andbody weight were similar to those of the control mice(not shown). Development of skin lesions was halted andin some cases even slightly reversed by IFN- + (Table 5).

To confirm, complement and extend further the results oflate IFN- + therapy in MRL-lpr mice, another experimentwas performed where MRL-lpr mice of intermediate age(e.g. 12 weeks) were treated three times weekly on alter-nate days until the age of 25 weeks with either 100 000 Urat IFN- + (n = 27) or PBS (n = 14). Exogenous IFN- + againaccelerated both the tempo and the cumulative inci-dence of mortality in MRL-lpr mice, 20/27 (74.1 %) of thetreated mice being dead within this period of time (meanage of death ± SD = 108.3 ± 9.7 days) as compared to8/14 (57.1 %) of the PBS-treated control mice whosemean age at death was 108.3 ± 9.7 days [p = 0.0261 vs.PBS-treated mice by Logrank (Mantel-Cox)].

3 Discussion

Our data demonstrate that exogenously administeredmouse/rat IFN- + prevented clinical signs of the SLE-likesyndrome when administered under an early prophylac-tic regimen and accelerated disease development whengiven therapeutically to SLE-affected animals.

The dichotomic effect of IFN- + in this model resemblesthat found in studies using models of adjuvant-inducedarthritis [19], type II collagen-induced arthritis (CIA) [20]and experimental autoimmune neuritis (EAN) [21] andaccords with the pleiotropic effects of IFN- + on theimmune system, where it can exert both pro- and anti-inflammatory properties. Here, the inhibitory effects ofIFN- + on Ab production [22], monocyte chemotaxis [23]and lymphoproliferation [24] might explain the dimin-ished serum titers of autoantibodies and circulatingIgG1, IgG2a and IgG3, the prevention of nephritic andpulmonary lesions and the reduced splenomegaly andlymphoproliferation of MRL-lpr mice treated prophylacti-cally with IFN- + .

The significant reduction of body weight in IFN- + -treatedMRL-lpr mice is noteworthy as MRL-lpr mice gain abnor-mal body weight because of massive lymphoprolifera-tion and liquid retention due to kidney dysfunction. Boththe general appearance of the IFN- + -treated mice andthe observation that a treatment regimen capable ofabolishing early mortality in MRL-lpr mice did not reducebody weight in sex- and age-matched CBA/J and NODmice mice suggests that this phenomenon is attributableto the beneficial effect of IFN- + and not to IFN- + toxicity.

In contrast to the above findings, therapeutically admin-istered IFN- + exacerbated disease development in MRL-lpr mice; the treated mice died more rapidly than thecontrol animals and exhibited augmented proteinuriaand more aggressive nephritis, but not pulmonitis, thanthe control animals. That the kidney, but not the lung,was one of the targets on which IFN- + exerted its pro-inflammatory activities might accord with the capacity ofIFN- + to stimulate the production of nephritogenic IgG3Ab [11], which were augmented in mice treated thera-peutically with IFN- + . In addition, IFN- + stimulates mac-rophages to release pro-inflammatory nephritogenicmediators such as IL-1, TNF- § and nitric oxide [2, 25].

The ultimate mechanism(s) determining whether IFN- +exerts anti- or pro-inflammatory properties remains to bedelineated. Although the existence of two compartmentsof IFN- + has been postulated, the systemic and intralesi-onal, which are endowed with anti-inflammatory andpro-inflammatory properties, respectively [26], it is diffi-cult to understand why, in MRL-lpr mice, exogenouslyadministered IFN- + should increase its systemic com-partment when given prophylactically and the intralesio-nal when applied therapeutically. The discrepant effectsof early vs. late treatment of MRL-lpr mice with IFN- +could also be explained on the basis of recent studies bySchwarting et al. [27] demonstrating that endogenousIFN- + limits macrophage expansion in an acceleratedform of autoimmune interstitial nephritis that can beinduced in MRL-lpr mice by gene transfer of macro-phage growth factors (CSF-1, GM-CSF-1) under therenal capsule. Accordingly, in preproteinuric mice earlyIFN- + prophylaxis might have counteracted CSF-1-induced recruitment of macrophages in the glomeruli,perhaps by inhibiting C3a expression [23]. Later on,when IFN- + treatment was started in mice affected byadvanced nephritis, IFN- + could no longer prevent mac-rophage influx and would rather activate them in the glo-meruli, leading to recruitment and stimulation of othercellular and humoral immune effectors.

An interference with the endogenous cytokine networkmay have also played a role in mediating the disease-modulating effects of exogenous IFN- + in MRL-lpr mice.If IFN- + exerted its well-known antagonistic functions onTh2 cells, two distinct pathogenic pathways could beenvisaged to operate in MRL-lpr mice, with Th2 cellsprevailing early during disease development in youngmice and Th1 cells becoming implicated later on duringthe course of the disease. Fluctuation of Th1 and Th2cells has been observed during the course of adjuvant-induced arthritis [19], type II CIA [28] and HgCl2-inducedsystemic autoimmunity in Brown Norway rats [29].Whether or not Th1 and Th2 cells alternatively interveneat different pathogenetic stages during the course of


SLE-like syndrome in Mrl-lpr mice is not known. How-ever, Yokoyama et al. [30] have observed a biphasicincrease in circulating and renal TNF- § in MRL-lpr mice,with an initial peak in neonatal mice which normalized by2 months of age and re-ascended proportionally to theseverity of renal injury. Since TNF- § is a type 1 pro-inflammatory cytokine, these data may concur with thepossibility that its presence or absence in sera and kid-neys of MRL-lpr mice at different ages mirrors alternateemergence and dampening of type 1 and type 2 cyto-kine responses, respectively, during the life-span ofMRL-lpr mice. However, since studies on the cytokineprofile of IFN- + -treated MRL-lpr mice have not yet beenperformed, one should be careful to attribute theobserved effects of IFN- + to a simple modulation of Th1/Th2 cells. Even though IFN- + antagonizes the action ofTh2-mediated events, under certain circumstances itmight also stimulate Th2 cell function, as it does in micewith experimentally induced SLE [12]. That IFN- + pro-phylaxis decreased circulating levels of both “type 1”(IgG2a, IgG3) and “type 2” (IgG1) cytokine Ig isotypeaccords with a marked, and previously described [22],inhibition of B cell function by IFN- + that is unrelated to aprevalence of Th1- or Th2-mediated B cell help. In con-trast, that the serum concentrations of classical “type 1cytokine Ig” such as IgG2a and nephritogenic IgG3 wereaugmented by the therapeutic treatment suggests thatunder these conditions exogenous IFN- + might haveenforced type 1 cytokine responses, and that this mighthave been associated with the deleterious effectsobserved. Here again, what dictates the different cyto-kine responses of exogenous IFN- + in young, disease-free MRL-lpr mice vs. SLE-affected animals is notknown.

How is it possible to reconcile the apparently paradoxi-cal protection by early IFN- + prophylaxis in MRL-lpr micewith the markedly diminished severity of the diseaseobserved both in IFN- + KO and IFN- + R-deficient MRL-lpr mice [14–16], and, along this vein, with the inefficacyof anti-IFN- + mAb to influence the course of the diseaseof these mice [7–8]? The effects of exogenously admin-istered cytokines do not necessarily mirror to the roleplayed by the endogenous cytokine in development ofboth physiological and pathological responses. In in vivostudies, blockade of the endogenous cytokine with spe-cific antagonists (mAb, soluble receptors) often exertsdifferent effects than the exogenous administration ofthe cytokine itself. For example, in spite of the preventiveefficacy of anti-IFN- + Ab in both NOD mice and BB ratinsulin-dependent diabetes mellitus (IDDM) [3], exoge-nously administered IFN- + either has no effects (NODmice) [3, 18] or paradoxically prevents disease develop-ment in BB rats [3]. On the other hand, mice with geneticcytokine deficiency may suffer from an abnormal matu-

ration of the immune system that may lead to activationof compensatory and regulatory mechanisms probablyless pronounced than those induced by prolonged treat-ment with exogenous cytokine or cytokine antagonist inadult mice with an intact (either normal or pathological)immune system. Thus, murine type II CIA may be exacer-bated by exogenous IFN- + [20] and still IFN- + R defi-ciency augments the susceptibility to it [31–32]. In a sim-ilar manner, while anti-IFN- + Ab prevent IDDM develop-ment in NOD mice [3], still IFN- + KO NOD mice developinsulitis and diabetes [3].

The dichotomic effects of exogenously administeredIFN- + in this classical model of human SLE are at vari-ance with the data obtained in the SLE-prone(NZBxNZW)F1 mice: irrespective of whether the treat-ment with IFN- + was started as early as at 2.5 months ordelayed until 6 months of age, it only exhibited pro-inflammatory effects, enhanced autoantibody produc-tion, aggravated glomerulonephritis development andaccelerated the rate of mortality of these mice [5]. Thus,whilst these data offer new insights into the compleximmunoregulatory effects of IFN- + and further revealpathogenic differences between the MRL-lpr and the(NZBxNZW)F1 models, their potential implications forthe clinical setting are unknown, especially becausetreatment with IFN- + has been associated with inductionof SLE [8].

Finally, the beneficial effects of both early and late treat-ment with IFN- + of the skin lesions of MRL-lpr mice con-cur with recent observations from Le Hir et al. [33] whoreported on a syndrome resembling human systemicsclerosis in MRL-lpr mice lacking IFN- + R. This devel-oped with severe skin lesions and histopathologicalcharacteristics of human scleroderma including mono-nuclear cell infiltrates and excessive production of colla-gen in skin, lungs, kidneys, liver, salivary glands andheart. Our present study supports this protective role forendogenous IFN- + in the development of skin lesions inMRL-lpr mice and demonstrates that exogenous IFN- +prevents them. Besides the potential clinical relevance ofthis observation for the treatment of human patients withsystemic sclerosis, where IFN- + has been shown to exertsome favorable effect [34], these data underscore furtherthat the immunopathogenic mechanisms operating inthe development of the SLE-like syndrome in MRL-lprmice are heterogeneous and that events that are patho-genetic in one organ might be beneficial in another.Along the same line, IFN- + may have both disease-preventing and -promoting effects on pathological mani-festations that affect different organs.


4 Materials and methods

4.1 Animals

Female MRL-lpr mice were purchased from Jackson Labo-ratory (Bar Harbor, ME) and kept under standard laboratoryconditions (non-SPF) with free access to food and water.Female CBA/J mice and diabetes-prone NOD mice micewere purchased from Charles River (Calco, Italy).

4.2 Rat and mouse IFN- q

Mouse rIFN- + was expressed in Escherichia coli and purifiedaccording to Gray and Goeddel [35]. Rat IFN- + was pro-duced by a CHO cell line transformed with a vector express-ing the chromosomal gene encoding rat IFN- + as describedelsewhere [36]. Endotoxin contamination of both IFN- + prep-arations was less than 80 pg/106 U as determined using theLimulus amebocyte lysate assay. Mouse or rat IFN- + wereadministered i.p. between 10.00 and 12.00 a.m.

4.3 Assessment of disease severity

Lymph node swelling was graded on a scale from 0 to 4 byan observer unaware of the treatment of the mice as follows:0 = none; 1 = barely palpable; 2 = large and palpable; 3 =visible and less than 0.5 cm2; 4 = visible and more than0.5 cm2. Skin lesions were assessed on a similar scale: 0 =none; 1 = less than 0.5 cm2 and localized to the head; 2 =0.5 to 1.5 cm2 and localized to the head; 3 = more than1.5 cm2; 4 = more than 2 cm2 extending to include the back.The mice were weighed once a week and examined for pro-teinuria by means of colorimetrical reaction using Albustix(Miles Laboratories, Elkhart, IN), graded as: trace (+/−)-= 10 mg/dl; (+) = 30 mg/dl; (++) = 100 mg/dl, (+++) = 300mg/dl and, (+++) = 1000 mg/dl. For statistical analysis theintensity of the colorimetrical reaction of each single mousewas reported numerically (10 mg/dl = 0.5, 30 mg/dl = 1,100 mg/dl = 2, 300 mg/dl = 3, and 1000 mg/dl = 4) and themean value from each experimental group calculated bydividing the total score by the number of mice in that group.Azotemia was was measured using Azostix (Ames, Elkhart,IN).

4.4 Measurement of serum Ig

Circulating IgG1, IgG2a, and IgG3 were measured by solid-phase ELISA, exactly as described by Takahashi et al. [11].Results are expressed in mg/ml in reference to a standardcurve obtained with mouse Ig reference serum.

4.5 Anti-ds DNA and -ss DNA Ab

Anti-ds- and anti-ss DNA Ab were studied by solid-phaseELISA using diluted serum samples (1/10, 1/100, 1/1000 inPBS-Tween 20) which were added in duplicate to microtiter

plates coated with human ds- or ss DNA (Hytech, Tel Aviv,Israel) as described in detail in a previous publication [6].

4.6 Histopathological study of the kidney

The histological signs of lupus nephritis were studied bylight microscopy examination of paraffin-embedded kidneysections by two pathologists unaware of the treatment of theanimals as described elsewhere [6, 7]. In brief, the extent ofthe pathological lesions was graded on a semiquantitativescale ranging from 0 to 4 as follows: 0 = normal; 1, a smallincrease of cells in the glomerular mesanguim; 2, a largernumber of cells in the mesangium; 3, glomerular lobular for-mation and thickened basement membrane; 4 glomerularcrescent formation, sclerosis, tubular atrophy and casts. Thescore for each animal was calculated by dividing the totalscore for the number of glomeruli observed.

4.7 Histopathological study of the lungs

Inflammatory pulmonary lesions were scored according toSeggev et al. [37]. Three vessels and three nonvascularareas in each of the five lobes were scored on a scale from0 to 3. Vessels were scored separately for (1) cells surround-ing the vessels (0, no surronding cells; 1, 1/3 of vessel sur-rounded; 2, 2/3 of vessel surrounded; 3, vessel completelyencircled by cells), and (2) infiltration of the vessel wall bycells (0, no infiltration; 1, 1/3 infiltrated; 2, 2/3 infiltrated; 3,complete infiltration). Therefore, the maximum number ofpoints for each vessel was 6. Nonvascular areas werescored for degree of infiltration (0, no infiltrate; 1, 10 infiltrat-ing mononuclear cells; 2, 20 infiltrating cells; 3, more than 20infiltrating cells). The maximum score for vascular areas was90 (6 × 3 × 5 lobes) and for interstitial areas 45 (3 × 3 × 5lobes). The perivascular and interstitial score were com-bined for each animal and the maximum possible score was135.

4.8 Immunofluorescence analysis

Spleens from individual mice were passes through sterilesieve and SLC obtained as described elsewhere [6]. SLC(106) were incubated with the appropriate concentration ofeither FITC- or PE-conjugated rat mAb (Pharmingen, SanDiego, CA) recognizing mouse T (Thy-1.2), B and NK cellsand the L3T4+ (CD4+), Ly-2 (CD8), B220+, CD45+ Ia+ andCD25+ subsets. Double immunofluorescence analysis wasperformed to evaluate the percentages of CD3+B220+ Tcells. Irrelevant rat IgG were used as control in each analy-sis. Cytofluorimetric analysis was performed using aFACScan (Becton Dickinson, Mountain View, CA) by anobserver unaware of the treatment of the mice.


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Correspondence: Ferdinando Nicoletti, Via Luigi Sturzo n.3,I-95021 Cannizzaro, Catania, ItalyFax: +39-095-325032e-mail: ferdinic — ctonline.it


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Dichotomic effects of IFN-γ on the development of systemic lupus erythematosus-like syndrome in MRL-lpr / lpr mice