INFECTION AND IMMUNITY, Sept. 1978, p. 925-9300019-9567/78/0021-0925$02.00/0Copyright © 1978 American Society for Microbiology

Vol. 21, No. 3

Printed in U.S.A.

Interferon Treatment of NZB Mice: Accelerated Progressionof Autoimmune Disease

H. HEREMANS,' * A. BILLIAU,' A. COLOMBATTI,L J. HILGERS,2 AND P. DE SOMER'

Department ofHuman Biology, Division ofMicrobiology, Rega Institute, University of Leuven, Leuven,Belgium, ' and Division of Genetics, The Netherlands Cancer Institute, Amsterdam, The Netherlands2

Received for publication 15 May 1978

The effect of long-term administration of interferon in New Zealand Black andNew Zealand Black/New Zealand White F, hybrid mice was studied. Treatmentwith moderate doses of interferon (104 units, five times weekly for 8 weeks) didnot depress marine leukemia virus gp69/71 levels in serum and spleen, nor p30levels in the spleen. Interferon given at 105' units (three times weekly for 37weeks) caused an increased incidence of anti-erythrocyte antibodies in NewZealand Black mice. Finally, the hybrid mice given interferon at 106" units (threetimes weekly for 33 weeks) had increased renal immune complex deposits andincreased incidences of proteinuria and anemia.

Systemic lupus erythematosus in humans isan autoimmune disease, the primary cause ofwhich is unknown. New Zealand Black (NZB)mice as well as their F1 hybrids with New Zea-land White mice (NZB/NZW-F,) spontaneouslydevelop an autoimmune disorder which in manyaspects resembles systemic lupus erythematosusin humans (7, 20). NZB mice develop hemolyticanemia; most animals die prematurely with se-vere anemia and hepatosplenomegaly. Some ofthe survivors develop chronic renal disease.NZB/NZW-F, mice develop a progressive andlethal immune complex type glomerulonephritisaccompanied by the appearance of circulatingantibodies reacting with nuclear material as wellas with DNA and RNA of various specificities(7, 9, 20).Two interacting factors, both ofwhich may be

genetically controlled, have been implicated inthe pathogenesis of the disease (7, 20): (i) faultycontrol of the immune system and (ii) formationof foreign antigens as a result of latent viralinfection. The virus involved would be a geneti-cally transmitted C-type oncornavirus (10, 12,13, 23). The replication of these viruses in chron-ically infected cell cultures is susceptible to theantiviral effect of interferon (2, 5). Therefore, itseemed possible that interferon administrationto NZB or NZB/NZW-F1 mice might delay orblock the progression of the autoimmune dis-ease.

MATERIALS AND METHODSExperimental animals. NZB mice were bred from

couples obtained from the Red Cross Blood Transfu-sion Service, Amsterdam, The Netherlands. NZB/NZW-F, hybrids were bred from female NZB mice

mated to NZW males obtained from the Instituut voorToegepast Natuurwetenschappelijk Onderzoek, Afdel-ing Proefdierbedrijf, Zeist, The Netherlands. NMRImice (Proefdiercentrum, University of Leuven, Bel-gium) were used for control experiments. Blood sam-ples for Coombs reactions and for hemoglobin andhematocrit determinations were taken from the tail;blood samples for determinations on serum were takenfrom the orbital sinus.

Interferon. Interferon and mock interferon wereprepared on mice L-929 cells. The cells were grown toconfluency in roller bottles, washed, refed with serum-free medium, and incubated at 370C for 24 h. Themedium was harvested and used as crude mock inter-feron. The cells were incubated with Newcastle diseasevirus at a multiplicity of infection of 1 plaque-formingunit per cell, incubated for 1 h, washed, refed withserum-free medium, and incubated for 24 h. The me-dium was harvested and used as crude interferon.Crude mock interferon and interferon were concen-trated and partially purified by fractional precipitationwith ammonium sulfate. Interferon assays were car-ried out on L-929 cells. Serial threefold dilutions (100Id per well) were made in duplicate in flat-bottommicrotiter plates (Falcon, Oxnard, Calif.). On eachplate a laboratory interferon standard consisting ofmouse L-929 cell interferon induced with Newcastledisease virus was included. To each well 100 ul of cellsuspension (6 x 10' cells per ml) was added. After 16h of incubation, the cultures were challenged with 501.d of Mengo virus (multiplicity of infection, 0.01plaque-forming unit per cell). When the cytopathiceffect was complete in virus controls, after 24 h ofincubation, the cells were washed with Dulbecco phos-phate-buffered saline (PBS) and stained with crystalviolet. All interferon doses in the present study areexpressed as research reference units per milliliter interms of the National Institutes of Health referencepreparation G-002-904-511. A concentration of 20 ref-erence units per ml was needed to provide 50% cyto-pathic effect protection in the assay described above.

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926 HEREMANS ET AL.

The interferon preparations had a specific activity inthe range of 10' to 107i' units per mg of protein.

Antisera. The following antisera were used: rabbitanti-mouse immunoglobulin (Ig) antibody, rabbit an-tiserum to mouse BC/BA globulin (C,), fluorescein-isothiocyanate (FITC)-labeled goat antiserum tomouse Ig, and FITC-labeled goat antiserum to rabbitIg. All antisera were purchased from Nordic Immu-nological Laboratory, Tilburg, The Netherlands.

Analytical methods. Proteinuria was determinedon fresh drops of urine, using tetrabromophenol paper(Albustix; Ames Co., Inc., Elkhart, Ind.). Reactions of3+ and 4+ (>1 mg/ml) were considered as significantproteinuria. Hematocrit values were determined witha standard capillary tube technique. Hemoglobin de-terminations were done on 20-LI samples of bloodusing the cyan-hematin method.

Anti-erythrocyte antibodies were determined by adirect Coombs reaction. The erythrocytes of eachmouse were washed twice in warm PBS without Ca`+and Mg2' at pH 7.4 and diluted to a 16% suspension inthe same buffer. A drop of this suspension was layeredon a glass plate and mixed with a drop of a suitabledilution of rabbit anti-mouse Ig antibody. Cells from10-months-old NZB mice and from young NMRI miceserved as positive and negative controls.

Determinations of p30 and gp69/71 were done byradioimmunoassay, using the interspecies determi-nants (11, 19). The reaction mixture for immunopre-cipitation contained the following: 0.003 ml of normalrabbit serum, 1 to 2 ng of '25I-labeled proteins (3 x 104to 6.5 x 104 cpm/ng), and 0.01 ml of diluted rabbitanti-feline virus (Theilen strain). The final volume wasadjusted to 0.2 ml with TEN buffer [20 mM tris-(hydroxymethyl)aminomethane-hydrochloride (pH7.6), 1 mM ethylenediaminetetraacetic acid, 100 mMNaCl] containing 2 mg of crystalline bovine serumalbumin per ml and Triton X-100 to a final concentra-tion of 0.2% in the assay. The mixture was incubatedfor 4 h at 37°C, after which 0.03 ml of goat anti-rabbitIgG was added to precipitate the antigen-antibodycomplexes. The mixture was incubated for 1 additionalh at 37°C and then overnight at 4°C. Cold TEN buffer(0.5 ml) was added, and the precipitate was collectedby centrifugation at 4°C. The pellet was washed twicewith 0.5 ml of cold TEN, and the '25I-labeled antigenpresent in the precipitate was measured in a gammacounter. The competition radioimmunoassay reactionmixture was the same except that the antiserum dilu-tion added was that which precipitated 50% of thelabeled antigen. Competing proteins (spleen extractsand serum) were diluted in TEN buffer containing 2mg of bovine serum albumin per ml and Triton X-100to a final concentration of 0.2%.

Anti-double-stranded DNA (dsDNA) antibodieswere detected by immunofluorescence on Crithidiaeluciliae (1) (kit supplied by the Central Laboratory ofthe Netherlands Red Cross Blood Transfusion Service,Amsterdam, The Netherlands). FITC-labeled goatanti-mouse Ig antibody was used.

Antinuclear antibody was detected by indirect im-munofluorescence on frozen sections (4 tm) of normalrat liver. The sections were incubated for 30 min atroom temperature with the mouse sera (1/10 dilutionin PBS), washed three times with Veronal buffer (pH

INFECT. IMMUN.

7.2), stained for 30 min with FITC-labeled goat anti-serum to mouse Ig, washed, and examined with afluorescence microscope equipped with a Ploem illu-mination system.Immune complex detection in kidneys. Fresh

kidney fragments were snap-frozen in isopentane, pre-cooled in liquid nitrogen. The specimens were kept at-70°C until examination. Unfixed cryostat sections (4,tm) were air-dried, incubated with the appropriateantiserum, and examined with a fluorescence micro-scope equipped with a Ploem illumination system. Fordetection of mouse Ig precipitates, the sections wereincubated for 30 min with a suitable dilution of FITC-labeled goat antiserum to mouse 1g. For detection ofC:, deposits, an indirect technique was used. The sec-tions were incubated for 30 min with a suitable dilutionof rabbit antiserum to mouse BC/BA globulin (C,).FITC-labeled goat antiserum to rabbit Ig was thenapplied for 30 min. The specificity and appropriatedilutions were tested using kidney sections of normalNMRI mice and old NZB/NZW-F, hybrids as negativeand positive controls.

RESULTS

Failure of interferon to reduce murineleukemia virus p30 and gp69/71 levels inNZB spleens. In a preliminary experiment, theeffect of interferon treatment on the levels ofthe major C-type viral protein p30 and of theviral envelope glycoprotein gp69/71 in serumand spleen was determined. Fifteen NZB mice(27 to 32 days old) were divided into three groupsreceiving either PBS, mock interferon, or inter-ferofh (104." units in 0.1 ml, intraperitoneally, fivetimes weekly) during 8 weeks. Radioimmunoas-say results for p30 and gp69/71 are shown inTable 1. In control mice, the p30 levels in thespleen and gp69/71 levels in the serum werecomparable to those reported for NZB mice inthe literature (19, 23); gp69/71 levels in thespleen were about twice as high, which might beaccounted for by a different method for prepa-ration of the spleen extract. Treatment withmock interferon or interferon did not influenceany of these levels.From these data it was concluded that exog-

enously administered interferon is unable to af-fect the production of the major protein of theendogenous NZB C-type virus, or at least thathigher dosages would be needed. Therefore, thedose of interferon was increased at least 10-foldin further experiments, in which the effect onthe progression of disease was evaluated.Accelerated progression of hemolytic

anemia in interferon-treated NZB mice. InNZB mice the main component of the autoim-mune disease is hemolytic anemia as manifestedby a positive Coombs reaction. The followingexperiment was done to study the effect of in-terferon on the progression ofthe disease. Thirty

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INTERFERON IN NZB MICE 927

NZB mice (15 males and 15 females; 29 to 36days old) were randomly divided in three groupsof 10. They were given PBS, mock interferon, orinterferon (105'3 units in 0.1 ml, intraperitoneally,three times weekly) during 37 weeks. Table 2shows spleen weights and incidences of anti-erythrocyte, antinuclear, and anti-dsDNA anti-bodies. The spleens of the female mice wereconsistently larger than those of the males. Bothin males and in females the mean spleen weightsof interferon-treated mice were higher thanthose of mice given PBS or mock interferon.However, all interferon-treated mice had alsoantierythrocyte antibodies, while more than 50%of the mice in both control groups were negativein this respect. Therefore, it seemed reasonableto conclude that, under the conditions of ourexperiment, interferon accelerated, rather thaninhibited, the progression of NZB disease. Theincidence of antinuclear and anti-dsDNA anti-body was low in all groups and seemed not to beaffected by the treatment.

TABLE 1. Effect of interferon on p30 and gp69/71levels in spleen and serum ofNZB mice

p30 pro- gp69/71 protein

Treatment" No: of tmice Spleen Spleen Serum

(ng/mg) (ng/mg) (jig/ml)PBS 4" 237.75 1,752.5 6.42

(35.08)' (442.66) (1.94)

Mock inter- 5 291.00 1,947.2 7.20feron (19.66) (540.29) (1.28)

Interferon 5 289.6 1,659.2 6.324(39.16) (607.86) (2.20)

"Groups of five NZB mice were injected intraperitoneallywith PBS, mock interferon, or interferon (10" units in 0.1 ml),five times weekly, during 8 weeks.

"One mouse died on day 56, due to unknown reasons.' Figures in parentheses are standard error of the mean.

Accelerated progression of renal diseasein interferon-treated NZB/NZW-F1 mice.To examine the effect of exogenous interferonon the development of renal autoimmune dis-ease, 30 female NZB/NZW-F, hybrid mice (28to 34 days old) were divided in three groups andwere given either PBS, mock interferon, or in-terferon (10';" units per 0.1 ml, intraperitoneally,three times weekly) for 33 weeks. Blood andurine samples were taken after 9, 13, 17, 22, and30 weeks and at the end of the experiment.Proteinuria, hemoglobin, hematocrit, and anti-bodies against erythrocytes, dsDNA, and nu-

clear DNA were determined. The kidneys were

examined for presence of immunoprecipitates.Body weights and terminal data for urine andblood are summarized in Table 3.

In each group the animals gained body weighttill the end of the experiment. The interferon-treated group was slightly behind on the twocontrol groups. Extensive edema, which mightexplain the gain in body weight, was not foundat autopsy. The spleen weights of interferon-treated mice were not increased as seen in NZBmice. The mean values of hematocrit and he-moglobin were slightly but not significantlylower in interferon-treated mice. At the time ofautopsy 1 of 10 PBS-treated mice, 2 of 10 mockinterferon-treated mice, and 4 of 9 interferon-treated mice had hematocrit values of s40%.Antierythrocyte antibodies were not detectedexcept in terminal serum samples. PositiveCoombs reactions occurred in two of nine inter-feron-treated mice and were completely absentin the two control groups. Taken together, thesefigures suggest an accelerated progression ofanemia in the interferon-treated group.

Antinuclear and anti-dsDNA antibodies were

only determined in the terminal serum samples.Positive reactions were randomly distributedover the three groups.

TABLE 2. Effect of interferon on hemolytic anemia in NZB miceMice No. of animals with:

Treatment" Mean spleen wt"Sexatme" No(mg) Anti-erythro- Antinuclear Anti-dsDNASex No. cyte antibody' antibody antibody

PBS Male 6 144 (20.64) 4 2 1Female 3 250 (52.68)

Mock interferon Male 4 83 (18.87) 4 2 0Female 5 333 (79.70)

Interferon Male 6 230 (60.26) 9 0 1Female 3 963 (452.6)

"Groups of 10 NZB mice were injected intraperitoneally with PBS, mock interferon, or interferon (10" unitsin 0.1 ml) three times weekly during 37 weeks. One animal of each group died during the experiment, due tounknown reasons.

" Analysis of variance (fixed effects model) was carried out according to Kendall (8); effect of sex: P < 0.01;effect of treatment: P < 0.05; interaction: not significant. Parentheses indicate standard error.

Difference between the interferon-treated group and the pooled PBS- and mock interferon-treated groupsis significant at the P < 0.02 level (X2 test with Yates correction).

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TABLE 3. Effect of interferon on the disease parameters of N7,B/NZW-F, hybrid mice

Treatment" No. of Body wt. () at age: Spleen wt % Erythro- Hemoglobinmmice 5 mo ; mo 7.5 mo 8.5 mo (mg) cytes (g per l(X) ml)

PBS 10 29.7 31.15 31.9 32.7 109 42.03 14.59(0.559)" (0.37) (0.393) (0.62) (13.61) (1.38) (0.48)

Mock interferon 10 27.55 30.25 31.25 31.55 93 44.26 15.11(0.639) (0.484) (0.716) (0.651) (8.119) (0.9) (0.19)

Interferon 9' 27.11 28.28 30.55 30.44 127 40.13 14.08(0.455) (0.434) (0.733) (0.757) (5.094) (1.66) (0.51)

No. of animals with:

Treatment" No. of Positive Proteinuria Renal fluorescencemice Coombs re- ANF' Anti-DNAaction -1 mg/ml >1 mg/ml Mild Severe

PBS 10 0 6 5 8 2 8 2Mock interferon 10 0 5 4 9 1 9 1Interferon 9' 2 7 3 5 4 5 4

" Groups of female NZB/NZW-F, mice were injected intraperitoneally with PBS, mock interferon, orinterferon (10" units in 0.1 ml) three times weekly during 33 weeks. All figures, except body weight, refer toterminal data.

"Figures in parentheses are standard error.'One moribund mouse was sacrificed on day 115; kidneys showed mild renal fluorescence, whereas the other

parameters were negative." ANF, Antinuclear antibody.

Proteinuria of >1 mg/ml (3+ or 4+) firstappeared in week 17 of the experiment. It ap-peared later (week 30) in the interferon-treatedmice. However, 4 of 9 animals in this group hadsevere proteinuria, against 1 of 10 and 2 of 10 inthe two control groups. This slight differenceprompted us to terminate the experiment toevaluate the kidneys immunologically. Fluores-cence, revealing antibodies to mouse Ig and C:I,was demonstrated in the glomeruli of all mice ineach group. It was graded in two patterns: (i)"mild" renal fluorescence with focal and lumpydeposits of Ig and C: preponderantly in themesangial spaces of the glomeruli and, in a fewinstances, sparsely along some capillary base-ment membranes (Fig. 1A), and (ii) "severe"renal fluorescence with coarse deposits of Ig andC: mainly in the glomerular capillary walls and,to a lesser extent, in the mesangium (Fig. 1B).The figures in Table 3 show that only a minority(3 of 20) of the mice in the control groups hadsevere fluorescence. In contrast, four of nineinterferon-treated mice had severe fluorescence.From these data it is clear that the interferontreatment certainly had no beneficial effect onthe development of the NZB/NZW autoimmunedisease. On the contrary, interferon-treated an-imals appeared to have slightly higher incidencerates of proteinuria, anemia, and severe renalfluorescence.Table 4 shows the correlation in the occur-

rence of the different disease parameters that

were measured. When all mice were consideredas one population, renal fluorescence, protein-uria, and low hematocrit and hemoglobin levelsoccurred in association with each other. Anti-nuclear and anti-dsDNA antibody seemed tooccur independently from these parameters.

DISCUSSIONLong-term treatment of the NZB with mod-

erate doses of interferon depressed neither thespleen levels of p30 and gp69/71 nor the serumlevels of gp69/71. Although the same doses ofinterferon were found to provide partial protec-tion against exogenous infection with Mengovirus (our unpublished data), the failure of in-terferon to depress p30 and gp69/71 levels couldbe anticipated. Indeed, studies with chronicallyinfected cell cultures have shown that the inter-feron-mediated inhibition of C-type virus repli-cation is not accompanied by a depression in thesynthesis of the major viral proteins (5, 17).Accordingly, it could also be predicted that in-terferon would not prevent NZB disease. How-ever, it was unexpected that long-term treat-ment with high doses of interferon acceleratedthe development of disease symptoms in NZBand NZB/NZW-F, mice. The mechanism of thisaccelerated development can at present only bespeculated upon. In fact, the effect may not evenbe due to interferon itself, but rather to impuri-ties in the preparation. With each injection ofinterferon, the mice also received small amounts

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FIG. 1. Fluorescent photomicrographs of renal glomeruli from 8.5-month-old NZB/NZW-F, mice treatedwith interferon. The preparations were stained with an FITC-labeled goat antiserum to mouse Ig. (x 400). (A)Mild fluorescence pattern. Ig is heavily deposited in the mesangial areas. (B) Severe fluorescence pattern. Igis deposited in the mesangium and along capillary wells. Comparable results occurred with antiserum tomouse C..

TABLE 4. Association of different NZB/NZW-F,disease parameters,with the extent of renal

fluorescenceRenal fluorescence

Parameters Significance"Severe Mild

Total 7"' 22Proteinuria (>1 mg/ 7 0 P < 0.(01

ml)Low hematocrit 6 2 P < 0.001

(G40%i)Low hemoglobin (614 7 0 P < 0.001mg/ml)

Antinuclear antibody 6 12 0.05 < P < 0.1Anti-dsDNA anti- 2 10 0.75 < P < 0.8body

"Statistical significance of association with severe renalfluorescence by X2 test with Yates correction.

" Number of mice in pooled groups (experiment shown inTable 3).

of foreign antigens, such as proteins from bovineserum or residual Newcastle disease virus com-ponents. The effect of these antigens on thedevelopment of disease symptoms was only par-tially controlled by the inclusion of a grouptreated with a mock interferon preparation: thispreparation did not contain Newcastle diseasevirus. The accelerated development of disease ininterferon-treated mice has some parallels in theliterature. Experimental infection with severalviruses was shown to result in earlier develop-ment of nephritis in NZB mice (14, 21). Theadministration of a synthetic double-strandedRNA, polyinosinic acid-polycytidylic acid, andTilorone, two potent interferon inducers, also

caused earlier disease symptoms in NZB mice(3, 18, 22). In contrast to these reports, a favor-able effect was observed using natural double-stranded RNA (16). Recently, it has been re-ported that interferon treatment of sucklingmice can lead to a progressive lethal glomerulo-nephritis in adult mice (6). Anti-interferon an-tibodies were shown to protect mice againstchronic disease caused by lymphocytic chorio-meningitis virus (15).Thus interferon, or molecules that are pro-

duced in association with interferon, might pro-voke rather than inhibit some of the symptomsof virus disease. A similar suggestion has comeLrom the observation that patients treated withinterferon develop malaise, fever, and transitorylymphopenia and that intracutaneous injectionof interferon elicits an inflammatory response(4).

ACKNOWLEDGMENTSThis work was supported by a grant from the Belgian

A.S.L.K. Cancer Research Foundation. The study on viralproteins was done during the tenure of an American CancerSociety-Eleanor Roosevelt-International Cancer Fellow-ship awarded by the International Union against cancer to A.C. on leave from the Institute of Pathological Anatomy, Uni-versity of Padou, Italy.We are indebted to B. Van Damme and E. Meulepas for

helpful discussions. Purified Rauscher murine leukemia virusp30 and gp69/71 and rabbit anti-feline leukemia virus serawere kindly provided by M. Strand, Albert Einstein College ofMedicine, New York. Anti-dsDNA determinations were doneby E. Stevens (Division of Immunology, A.Z. Pellenberg,Leuven). The technical assistance of Chris Neuckermans-Dil-len, Francine Cornette, Lieve Godefridus, R. Conings, R.Wijnants, and P. Moerkerk, and the editorial help of JaninePutzeys, are appreciated.

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