INFECTION AND IMMUNITY, Sept. 1978, p. 925-930 0019-9567/78/0021-0925$02.00/0 Copyright © 1978 American Society for Microbiology
Vol. 21, No. 3
Printed in U.S.A.
Interferon Treatment of NZB Mice: Accelerated Progression of Autoimmune Disease H. HEREMANS,' * A. BILLIAU,' A.
COLOMBATTI,L J. HILGERS,2
AND P.
DE SOMER'
Department of Human Biology, Division of Microbiology, 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 and New Zealand Black/New Zealand White F, hybrid mice was studied. Treatment with moderate doses of interferon (104 units, five times weekly for 8 weeks) did not depress marine leukemia virus gp69/71 levels in serum and spleen, nor p30 levels in the spleen. Interferon given at 105' units (three times weekly for 37 weeks) caused an increased incidence of anti-erythrocyte antibodies in New Zealand Black mice. Finally, the hybrid mice given interferon at 106" units (three times weekly for 33 weeks) had increased renal immune complex deposits and increased incidences of proteinuria and anemia.
Systemic lupus erythematosus in humans is mated to NZW males obtained from the Instituut voor an autoimmune disease, the primary cause of Toegepast Natuurwetenschappelijk Onderzoek, Afdelwhich is unknown. New Zealand Black (NZB) ing Proefdierbedrijf, Zeist, The Netherlands. NMRI mice (Proefdiercentrum, University of Leuven, Belmice as well as their F1 hybrids with New Zea- gium) used for control experiments. Blood samland White mice (NZB/NZW-F,) spontaneously ples forwere Coombs reactions and for hemoglobin and develop an autoimmune disorder which in many hematocrit determinations were taken from the tail; aspects resembles systemic lupus erythematosus blood samples for determinations on serum were taken in humans (7, 20). NZB mice develop hemolytic from the orbital sinus. anemia; most animals die prematurely with seInterferon. Interferon and mock interferon were vere anemia and hepatosplenomegaly. Some of prepared on mice L-929 cells. The cells were grown to the survivors develop chronic renal disease. confluency in roller bottles, washed, refed with serumNZB/NZW-F, mice develop a progressive and free medium, and incubated at 370C for 24 h. The medium was harvested and used as crude mock interlethal immune complex type glomerulonephritis feron. cells were incubated with Newcastle disease accompanied by the appearance of circulating virus atThe a multiplicity of infection of 1 plaque-forming antibodies reacting with nuclear material as well unit per cell, incubated for 1 h, washed, refed with as with DNA and RNA of various specificities serum-free medium, and incubated for 24 h. The me(7, 9, 20). dium was harvested and used as crude interferon. Two interacting factors, both of which may be Crude mock interferon and interferon were concengenetically controlled, have been implicated in trated and partially purified by fractional precipitation the pathogenesis of the disease (7, 20): (i) faulty with ammonium sulfate. Interferon assays were carcontrol of the immune system and (ii) formation ried out on L-929 cells. Serial threefold dilutions (100 of foreign antigens as a result of latent viral Id per well) were made in duplicate in flat-bottom plates (Falcon, Oxnard, Calif.). On each infection. The virus involved would be a geneti- microtiter a laboratory interferon standard consisting of cally transmitted C-type oncornavirus (10, 12, plate mouse L-929 cell interferon induced with Newcastle 13, 23). The replication of these viruses in chron- disease virus was included. To each well 100 ul of cell ically infected cell cultures is susceptible to the suspension (6 x 10' cells per ml) was added. After 16 antiviral effect of interferon (2, 5). Therefore, it h of incubation, the cultures were challenged with 50 seemed possible that interferon administration 1.d of Mengo virus (multiplicity of infection, 0.01 to NZB or NZB/NZW-F1 mice might delay or plaque-forming unit per cell). When the cytopathic block the progression of the autoimmune dis- effect was complete in virus controls, after 24 h of incubation, the cells were washed with Dulbecco phosease. phate-buffered saline (PBS) and stained with crystal violet. All interferon doses in the present study are MATERIALS AND METHODS expressed as research reference units per milliliter in Experimental animals. NZB mice were bred from terms of the National Institutes of Health reference couples obtained from the Red Cross Blood Transfu- preparation G-002-904-511. A concentration of 20 refsion Service, Amsterdam, The Netherlands. NZB/ erence units per ml was needed to provide 50% cytoNZW-F, hybrids were bred from female NZB mice pathic effect protection in the assay described above. 925
926
INFECT. IMMUN.
HEREMANS ET AL.
The interferon preparations had a specific activity in the range of 10' to 107i' units per mg of protein. Antisera. The following antisera were used: rabbit anti-mouse immunoglobulin (Ig) antibody, rabbit antiserum to mouse BC/BA globulin (C,), fluoresceinisothiocyanate (FITC)-labeled goat antiserum to mouse Ig, and FITC-labeled goat antiserum to rabbit Ig. All antisera were purchased from Nordic Immunological Laboratory, Tilburg, The Netherlands. Analytical methods. Proteinuria was determined on fresh drops of urine, using tetrabromophenol paper (Albustix; Ames Co., Inc., Elkhart, Ind.). Reactions of 3+ and 4+ (>1 mg/ml) were considered as significant proteinuria. Hematocrit values were determined with a standard capillary tube technique. Hemoglobin determinations were done on 20-LI samples of blood using the cyan-hematin method. Anti-erythrocyte antibodies were determined by a direct Coombs reaction. The erythrocytes of each mouse were washed twice in warm PBS without Ca`+ and Mg2' at pH 7.4 and diluted to a 16% suspension in the same buffer. A drop of this suspension was layered on a glass plate and mixed with a drop of a suitable dilution of rabbit anti-mouse Ig antibody. Cells from 10-months-old NZB mice and from young NMRI mice served as positive and negative controls. Determinations of p30 and gp69/71 were done by radioimmunoassay, using the interspecies determinants (11, 19). The reaction mixture for immunoprecipitation contained the following: 0.003 ml of normal rabbit serum, 1 to 2 ng of '25I-labeled proteins (3 x 104 to 6.5 x 104 cpm/ng), and 0.01 ml of diluted rabbit anti-feline virus (Theilen strain). The final volume was adjusted to 0.2 ml with TEN buffer [20 mM tris(hydroxymethyl)aminomethane-hydrochloride (pH 7.6), 1 mM ethylenediaminetetraacetic acid, 100 mM NaCl] containing 2 mg of crystalline bovine serum albumin per ml and Triton X-100 to a final concentration of 0.2% in the assay. The mixture was incubated for 4 h at 37°C, after which 0.03 ml of goat anti-rabbit IgG was added to precipitate the antigen-antibody complexes. The mixture was incubated for 1 additional h at 37°C and then overnight at 4°C. Cold TEN buffer (0.5 ml) was added, and the precipitate was collected by centrifugation at 4°C. The pellet was washed twice with 0.5 ml of cold TEN, and the '25I-labeled antigen present in the precipitate was measured in a gamma counter. The competition radioimmunoassay reaction mixture was the same except that the antiserum dilution added was that which precipitated 50% of the labeled antigen. Competing proteins (spleen extracts and serum) were diluted in TEN buffer containing 2 mg of bovine serum albumin per ml and Triton X-100 to a final concentration of 0.2%. Anti-double-stranded DNA (dsDNA) antibodies were detected by immunofluorescence on Crithidiae luciliae (1) (kit supplied by the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands). FITC-labeled goat anti-mouse Ig antibody was used. Antinuclear antibody was detected by indirect immunofluorescence on frozen sections (4 tm) of normal rat liver. The sections were incubated for 30 min at room temperature with the mouse sera (1/10 dilution in PBS), washed three times with Veronal buffer (pH
7.2), stained for 30 min with FITC-labeled goat antiserum to mouse Ig, washed, and examined with a fluorescence microscope equipped with a Ploem illumination system. Immune complex detection in kidneys. Fresh kidney fragments were snap-frozen in isopentane, precooled in liquid nitrogen. The specimens were kept at -70°C until examination. Unfixed cryostat sections (4 ,tm) were air-dried, incubated with the appropriate antiserum, and examined with a fluorescence microscope equipped with a Ploem illumination system. For detection of mouse Ig precipitates, the sections were incubated for 30 min with a suitable dilution of FITClabeled goat antiserum to mouse 1g. For detection of C:, deposits, an indirect technique was used. The sections were incubated for 30 min with a suitable dilution of rabbit antiserum to mouse BC/BA globulin (C,). FITC-labeled goat antiserum to rabbit Ig was then applied for 30 min. The specificity and appropriate dilutions were tested using kidney sections of normal NMRI mice and old NZB/NZW-F, hybrids as negative and positive controls.
RESULTS Failure of interferon to reduce murine leukemia virus p30 and gp69/71 levels in NZB spleens. In a preliminary experiment, the effect of interferon treatment on the levels of the major C-type viral protein p30 and of the viral envelope glycoprotein gp69/71 in serum and spleen was determined. Fifteen NZB mice (27 to 32 days old) were divided into three groups receiving either PBS, mock interferon, or interferofh (104." units in 0.1 ml, intraperitoneally, five times weekly) during 8 weeks. Radioimmunoassay results for p30 and gp69/71 are shown in Table 1. In control mice, the p30 levels in the spleen and gp69/71 levels in the serum were comparable to those reported for NZB mice in the literature (19, 23); gp69/71 levels in the spleen were about twice as high, which might be accounted for by a different method for preparation of the spleen extract. Treatment with mock interferon or interferon did not influence any of these levels. From these data it was concluded that exogenously administered interferon is unable to affect the production of the major protein of the endogenous NZB C-type virus, or at least that higher dosages would be needed. Therefore, the dose of interferon was increased at least 10-fold in further experiments, in which the effect on the progression of disease was evaluated. Accelerated progression of hemolytic anemia in interferon-treated NZB mice. In NZB mice the main component of the autoimmune disease is hemolytic anemia as manifested by a positive Coombs reaction. The following experiment was done to study the effect of interferon on the progression of the disease. Thirty
INTERFERON IN NZB MICE
VOL. 21, 1978
NZB mice (15 males and 15 females; 29 to 36 days old) were randomly divided in three groups of 10. They were given PBS, mock interferon, or interferon (105'3 units in 0.1 ml, intraperitoneally, three times weekly) during 37 weeks. Table 2 shows spleen weights and incidences of antierythrocyte, antinuclear, and anti-dsDNA antibodies. The spleens of the female mice were consistently larger than those of the males. Both in males and in females the mean spleen weights of interferon-treated mice were higher than those of mice given PBS or mock interferon. However, all interferon-treated mice had also antierythrocyte antibodies, while more than 50% of the mice in both control groups were negative in this respect. Therefore, it seemed reasonable to conclude that, under the conditions of our experiment, interferon accelerated, rather than inhibited, the progression of NZB disease. The incidence of antinuclear and anti-dsDNA antibody was low in all groups and seemed not to be affected by the treatment. TABLE 1. Effect of interferon on p30 and gp69/71 levels in spleen and serum of NZB mice p30 proTreatment"
No: miceof 4"
PBS
gp69/71 protein
t Spleen
Spleen
Serum
(ng/mg)
(ng/mg)
(jig/ml)
(35.08)'
1,752.5 (442.66)
6.42 (1.94)
237.75
Mock interferon
5
291.00 (19.66)
1,947.2 (540.29)
7.20 (1.28)
Interferon
5
289.6 (39.16)
1,659.2
6.324
(607.86) (2.20) "Groups of five NZB mice were injected intraperitoneally with 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.
927
Accelerated progression of renal disease in interferon-treated NZB/NZW-F1 mice. To examine the effect of exogenous interferon on the development of renal autoimmune disease, 30 female NZB/NZW-F, hybrid mice (28 to 34 days old) were divided in three groups and were given either PBS, mock interferon, or interferon (10';" units per 0.1 ml, intraperitoneally, three times weekly) for 33 weeks. Blood and urine samples were taken after 9, 13, 17, 22, and 30 weeks and at the end of the experiment. Proteinuria, hemoglobin, hematocrit, and antibodies against erythrocytes, dsDNA, and nuclear DNA were determined. The kidneys were examined for presence of immunoprecipitates. Body weights and terminal data for urine and blood are summarized in Table 3. In each group the animals gained body weight till the end of the experiment. The interferontreated group was slightly behind on the two control groups. Extensive edema, which might explain the gain in body weight, was not found at autopsy. The spleen weights of interferontreated mice were not increased as seen in NZB mice. The mean values of hematocrit and hemoglobin were slightly but not significantly lower in interferon-treated mice. At the time of autopsy 1 of 10 PBS-treated mice, 2 of 10 mock interferon-treated mice, and 4 of 9 interferontreated mice had hematocrit values of s40%. Antierythrocyte antibodies were not detected except in terminal serum samples. Positive Coombs reactions occurred in two of nine interferon-treated mice and were completely absent in the two control groups. Taken together, these figures suggest an accelerated progression of anemia in the interferon-treated group. Antinuclear and anti-dsDNA antibodies were only determined in the terminal serum samples. Positive reactions were randomly distributed over the three groups.
TABLE 2. Effect of interferon on hemolytic anemia in NZB mice No. of animals with:
Mice Treatment"
Sexatme" Sex
Mean spleen wt"
No(mg) No.
Anti-erythro-
cyte antibody'
Antinuclear antibody
Anti-dsDNA antibody
1 2 4 6 144 (20.64) 3 250 (52.68) 0 2 4 83 (18.87) 4 Mock interferon Female 5 333 (79.70) 1 0 6 9 Male 230 (60.26) Interferon 3 Female 963 (452.6) "Groups of 10 NZB mice were injected intraperitoneally with PBS, mock interferon, or interferon (10" units in 0.1 ml) three times weekly during 37 weeks. One animal of each group died during the experiment, due to unknown 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 groups is significant at the P < 0.02 level (X2 test with Yates correction). PBS
Male Female Male
928
INFECT. IMMUN.
HEREMANS ET AL.
TABLE 3. Effect of interferon on the disease parameters of N7,B/NZW-F, hybrid mice Treatment"
No. of
mmice
PBS
5 mo
10
Mock interferon Interferon
Body wt. () at age: ; mo mo
7.5
8.5 mo
Spleen wt (mg)
% Erythrocytes
Hemoglobin
(g per l(X) ml)
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)
93
(8.119)
44.26 (0.9)
(0.19)
10
27.55
30.25
31.25
(0.639)
(0.484)
(0.716)
31.55 (0.651)
27.11
28.28 (0.434)
30.55 (0.733)
30.44
127
40.13
(0.757)
(5.094)
(1.66)
9'
(0.455)
15.11
14.08 (0.51)
No. of animals with: Treatment"
of No. mice
PositivereCoombs action
ANF'
Anti-DNA
Proteinuria -1 mg/ml
>1 mg/ml
Renal fluorescence Mild
Severe
10 0 6 PBS 5 8 2 8 2 0 10 Mock interferon 5 4 9 1 9 1 9' 2 7 Interferon 3 5 4 5 4 " Groups of female NZB/NZW-F, mice were injected intraperitoneally with PBS, mock interferon, or interferon (10" units in 0.1 ml) three times weekly during 33 weeks. All figures, except body weight, refer to terminal 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+) first appeared in week 17 of the experiment. It appeared later (week 30) in the interferon-treated mice. However, 4 of 9 animals in this group had severe proteinuria, against 1 of 10 and 2 of 10 in the two control groups. This slight difference prompted us to terminate the experiment to evaluate the kidneys immunologically. Fluorescence, revealing antibodies to mouse Ig and C:I, was demonstrated in the glomeruli of all mice in each group. It was graded in two patterns: (i) "mild" renal fluorescence with focal and lumpy deposits of Ig and C: preponderantly in the mesangial spaces of the glomeruli and, in a few instances, sparsely along some capillary basement membranes (Fig. 1A), and (ii) "severe" renal fluorescence with coarse deposits of Ig and C: 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 had severe fluorescence. In contrast, four of nine interferon-treated mice had severe fluorescence. From these data it is clear that the interferon treatment certainly had no beneficial effect on the development of the NZB/NZW autoimmune disease. On the contrary, interferon-treated animals appeared to have slightly higher incidence rates of proteinuria, anemia, and severe renal fluorescence. Table 4 shows the correlation in the occurrence of the different disease parameters that
were measured. When all mice were considered as one population, renal fluorescence, proteinuria, and low hematocrit and hemoglobin levels occurred in association with each other. Antinuclear and anti-dsDNA antibody seemed to occur independently from these parameters.
DISCUSSION Long-term treatment of the NZB with moderate doses of interferon depressed neither the spleen levels of p30 and gp69/71 nor the serum levels of gp69/71. Although the same doses of interferon were found to provide partial protection against exogenous infection with Mengo virus (our unpublished data), the failure of interferon to depress p30 and gp69/71 levels could be anticipated. Indeed, studies with chronically infected cell cultures have shown that the interferon-mediated inhibition of C-type virus replication is not accompanied by a depression in the synthesis of the major viral proteins (5, 17). Accordingly, it could also be predicted that interferon would not prevent NZB disease. However, it was unexpected that long-term treatment with high doses of interferon accelerated the development of disease symptoms in NZB and NZB/NZW-F, mice. The mechanism of this accelerated development can at present only be speculated upon. In fact, the effect may not even be due to interferon itself, but rather to impurities in the preparation. With each injection of interferon, the mice also received small amounts
VOL. 21, 1978
INTERFERON IN NZB MICE
929
FIG. 1. Fluorescent photomicrographs of renal glomeruli from 8.5-month-old NZB/NZW-F, mice treated with 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. Ig is deposited in the mesangium and along capillary wells. Comparable results occurred with antiserum to mouse C.. TABLE 4. Association of different NZB/NZW-F, disease parameters,with the extent of renal
fluorescence Renal fluorescence
Parameters Total Proteinuria (>1 mg/
Significance" Severe 7"'
Mild 22
0 7 P < 0.(01 ml) 6 Low hematocrit 2 P < 0.001 (G40%i) 7 Low hemoglobin (614 0 P < 0.001 mg/ml) 6 Antinuclear antibody 12 0.05 < P < 0.1 Anti-dsDNA anti2 10 0.75 < P < 0.8 body "Statistical significance of association with severe renal fluorescence by X2 test with Yates correction. " Number of mice in pooled groups (experiment shown in
Table 3).
of foreign antigens, such as proteins from bovine serum or residual Newcastle disease virus components. The effect of these antigens on the development of disease symptoms was only partially controlled by the inclusion of a group treated with a mock interferon preparation: this preparation did not contain Newcastle disease virus. The accelerated development of disease in interferon-treated mice has some parallels in the literature. Experimental infection with several viruses was shown to result in earlier development of nephritis in NZB mice (14, 21). The administration of a synthetic double-stranded RNA, polyinosinic acid-polycytidylic acid, and Tilorone, two potent interferon inducers, also
caused earlier disease symptoms in NZB mice (3, 18, 22). In contrast to these reports, a favorable effect was observed using natural doublestranded RNA (16). Recently, it has been reported that interferon treatment of suckling mice can lead to a progressive lethal glomerulonephritis in adult mice (6). Anti-interferon antibodies were shown to protect mice against chronic disease caused by lymphocytic choriomeningitis virus (15). Thus interferon, or molecules that are produced in association with interferon, might provoke rather than inhibit some of the symptoms of virus disease. A similar suggestion has come Lrom the observation that patients treated with interferon develop malaise, fever, and transitory lymphopenia and that intracutaneous injection of interferon elicits an inflammatory response
(4). ACKNOWLEDGMENTS This work was supported by a grant from the Belgian A.S.L.K. Cancer Research Foundation. The study on viral proteins was done during the tenure of an American Cancer Society-Eleanor Roosevelt-International Cancer Fellowship awarded by the International Union against cancer to A. C. on leave from the Institute of Pathological Anatomy, University of Padou, Italy. We are indebted to B. Van Damme and E. Meulepas for helpful discussions. Purified Rauscher murine leukemia virus p30 and gp69/71 and rabbit anti-feline leukemia virus sera were kindly provided by M. Strand, Albert Einstein College of Medicine, New York. Anti-dsDNA determinations were done by E. Stevens (Division of Immunology, A.Z. Pellenberg, Leuven). The technical assistance of Chris Neuckermans-Dillen, Francine Cornette, Lieve Godefridus, R. Conings, R. Wijnants, and P. Moerkerk, and the editorial help of Janine Putzeys, are appreciated.
930
HEREMANS ET AL. LITERATURE CITED
1. Aarden, L. A., E. R. De Groot, and T. E. W. Feltkamp. 1975. Immunology of DNA. III. Crithidia luciliae. A simple substrate for the determination of anti-dsDNA with the immunofluorescence technique. Ann. N.Y. Acad. Sci. 254:505-515. 2. Billiau, A., H. Sobis, and P. De Somer. 1973. Influence of interferon on virus particle formation in different oncornavirus carrier cell lines. Int. J. Cancer 12:646-653. 3. Carpenter, D. F., A. D. Steinberg, P. H. Schur, and N. Talal. 1970. The pathogenesis of autoimmunity in New Zealand mice. II. Acceleration of glomerulonephritis by polyinosinic polycytidylic acid. Lab. Invest. 23:628-(634. 4. De Somer, P., V. G. Edy, and A. Billiau. 1977. Interferon-induced skin reactivity in man. Lancet ii:47-48. 5. Friedman, R. M., E. H. Chang, J. M. Ramseur, and M. W. Myers. 1975. Interferon-directed inhibition of chronic murine leukemia virus production in cell cultures: lack of effect on intracellular viral markers. J. Virol. 16:569-574. 6. Gresser, I., C. Maury, M. Tovey, L. Morel-Maroger, and F. Pontillon. 1976. Progressive glomerulonephritis in mice treated with interferon preparations at birth. Nature (London) 263:420-422. 7. Howie, J. B., and L. D. Simpson. 1974. The immunopathology of the NZB mice and their hybrids, p. 116-163. In P. Miescher and H. Muller-Eberhard (ed.), Textbook of immunopathology. Greene and Stratton Publishers, New York. 8. Kendall, M. G. 1948. The advanced theory of statistics, vol. II, 2nd ed., p. 220-228. Ch. Griffins Co., Ltd., London. 9. Lambert, P. H., and F. J. Dixon. 1968. Pathogenesis of the glomerulonephritis of NZB/W mice. J. Exp. Med. 127:5(07-522. 10. Levy, J. A., P. Kazan, 0. Varnier, and A. Kleiman. 1975. Murine xenotropic type C virus. I. Distribution and further characterization of the virus in NZB mice. J. Virol. 16:844-853. 11. Melief, C. J. M., S. Louie, and R. S. Schwartz. 1975. Ecotropic leukemic viruses in congenic C57BL mice: natural dissemination by milk-borne infections. J. Natd. Cancer Inst. 55:691-698.
INFECT. IMMUN. 12. Mellors, R. C., T. Aoki, and R. J. Huebner. 1969. Further implications of murine leukemia-like virus in the disorders of NZB mice. J. Exp. Med. 129:1045-1062. 1;3. Mellors, R. C., T. Shirai, T. Aoki, R. J. Huebner, and K. Krawczywski. 1971. Wild-type Gross leukemia virus and the pathogenesis of the glomerulonephritis of New Zealand mice. J. Exp. Med. 133:113-132. 14. Oldstone, M. B. A. 1975. Virus neutralization and virusinduced immune complex disease. Progr. Med. Virol. 19:84-119. 15. Riviere, Y., I. Gresser, J. C. Guillon, and M. G. Tovey. 1977. Inhibition by anti-interferon serum of lymphocytic choriomeningitis virus disease in suckling mice. Proc. Natl. Acad. Sci. U.S.A. 74:2135-21:39. 16. Rovensky, J., J. Doskoch, J. Pekarck, L. Borecky, D. Zitnan, and J. Svejcar. 1975. Prevention of spontaneous autoimmunity to DNA in NZB/Swiss mice by treatment with natural double-stranded RNA. Immunology 29:745-748. 17. Shapiro, S. Z., M. Strand, and A. Billiau. 1977. Synthesis and cleavage processing of oncornavirus proteins during interferon inhibition of virus particle release. Infect. Immun. 16:742-747. 18. Steinberg, A. D., S. Baron, and N. Talal. 1969. The pathogenesis of autoimmunity in New Zealand Black mice. I. Induction of antinucleic acid antibodies by polyinosinic polycytidylic acid. Proc. Natl. Acad. Sci. U.S.A. 63:1102-11(7. 19. Strand, M., and J. T. August. 1976. Oncornavirus envelope glycoprotein in serom of' mice. Virology 75:130-144. 20. Talal, N., and A. D. Steinberg. 1974. The pathogenesis of autoimmunity in New Zealand Black mice. Curr. Top. Microbiol. Immunol. 64:79-103. 21. Tonietti, G., M. B. Oldstone, and F. J. Dixon. 1970. The effects of induced chronic viral infections on the immunological disease of New Zealand mice. ,J. Exp. Med. 132:89-1(09. 22. Walker, S. E. 1977. Accelerated mortality in NZB/NZW mice treated with the interferon inducer tilorone hydrochloride. Clin. Immunol. Immunopathol. 8:204-212. 23. Yoshiki, G., R. C. Mellors, M. Strand, and J. T. August. 1974. The viral envelope glycoprotein of murine leukemia virus and the pathogenesis of immune complex glomerulonephritis of New Zealand mice. J. Exp. Med. 140:1011-1027.
Vol. 21, No. 3
Printed in U.S.A.
Interferon Treatment of NZB Mice: Accelerated Progression of Autoimmune Disease H. HEREMANS,' * A. BILLIAU,' A.
COLOMBATTI,L J. HILGERS,2
AND P.
DE SOMER'
Department of Human Biology, Division of Microbiology, 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 and New Zealand Black/New Zealand White F, hybrid mice was studied. Treatment with moderate doses of interferon (104 units, five times weekly for 8 weeks) did not depress marine leukemia virus gp69/71 levels in serum and spleen, nor p30 levels in the spleen. Interferon given at 105' units (three times weekly for 37 weeks) caused an increased incidence of anti-erythrocyte antibodies in New Zealand Black mice. Finally, the hybrid mice given interferon at 106" units (three times weekly for 33 weeks) had increased renal immune complex deposits and increased incidences of proteinuria and anemia.
Systemic lupus erythematosus in humans is mated to NZW males obtained from the Instituut voor an autoimmune disease, the primary cause of Toegepast Natuurwetenschappelijk Onderzoek, Afdelwhich is unknown. New Zealand Black (NZB) ing Proefdierbedrijf, Zeist, The Netherlands. NMRI mice (Proefdiercentrum, University of Leuven, Belmice as well as their F1 hybrids with New Zea- gium) used for control experiments. Blood samland White mice (NZB/NZW-F,) spontaneously ples forwere Coombs reactions and for hemoglobin and develop an autoimmune disorder which in many hematocrit determinations were taken from the tail; aspects resembles systemic lupus erythematosus blood samples for determinations on serum were taken in humans (7, 20). NZB mice develop hemolytic from the orbital sinus. anemia; most animals die prematurely with seInterferon. Interferon and mock interferon were vere anemia and hepatosplenomegaly. Some of prepared on mice L-929 cells. The cells were grown to the survivors develop chronic renal disease. confluency in roller bottles, washed, refed with serumNZB/NZW-F, mice develop a progressive and free medium, and incubated at 370C for 24 h. The medium was harvested and used as crude mock interlethal immune complex type glomerulonephritis feron. cells were incubated with Newcastle disease accompanied by the appearance of circulating virus atThe a multiplicity of infection of 1 plaque-forming antibodies reacting with nuclear material as well unit per cell, incubated for 1 h, washed, refed with as with DNA and RNA of various specificities serum-free medium, and incubated for 24 h. The me(7, 9, 20). dium was harvested and used as crude interferon. Two interacting factors, both of which may be Crude mock interferon and interferon were concengenetically controlled, have been implicated in trated and partially purified by fractional precipitation the pathogenesis of the disease (7, 20): (i) faulty with ammonium sulfate. Interferon assays were carcontrol of the immune system and (ii) formation ried out on L-929 cells. Serial threefold dilutions (100 of foreign antigens as a result of latent viral Id per well) were made in duplicate in flat-bottom plates (Falcon, Oxnard, Calif.). On each infection. The virus involved would be a geneti- microtiter a laboratory interferon standard consisting of cally transmitted C-type oncornavirus (10, 12, plate mouse L-929 cell interferon induced with Newcastle 13, 23). The replication of these viruses in chron- disease virus was included. To each well 100 ul of cell ically infected cell cultures is susceptible to the suspension (6 x 10' cells per ml) was added. After 16 antiviral effect of interferon (2, 5). Therefore, it h of incubation, the cultures were challenged with 50 seemed possible that interferon administration 1.d of Mengo virus (multiplicity of infection, 0.01 to NZB or NZB/NZW-F1 mice might delay or plaque-forming unit per cell). When the cytopathic block the progression of the autoimmune dis- effect was complete in virus controls, after 24 h of incubation, the cells were washed with Dulbecco phosease. phate-buffered saline (PBS) and stained with crystal violet. All interferon doses in the present study are MATERIALS AND METHODS expressed as research reference units per milliliter in Experimental animals. NZB mice were bred from terms of the National Institutes of Health reference couples obtained from the Red Cross Blood Transfu- preparation G-002-904-511. A concentration of 20 refsion Service, Amsterdam, The Netherlands. NZB/ erence units per ml was needed to provide 50% cytoNZW-F, hybrids were bred from female NZB mice pathic effect protection in the assay described above. 925
926
INFECT. IMMUN.
HEREMANS ET AL.
The interferon preparations had a specific activity in the range of 10' to 107i' units per mg of protein. Antisera. The following antisera were used: rabbit anti-mouse immunoglobulin (Ig) antibody, rabbit antiserum to mouse BC/BA globulin (C,), fluoresceinisothiocyanate (FITC)-labeled goat antiserum to mouse Ig, and FITC-labeled goat antiserum to rabbit Ig. All antisera were purchased from Nordic Immunological Laboratory, Tilburg, The Netherlands. Analytical methods. Proteinuria was determined on fresh drops of urine, using tetrabromophenol paper (Albustix; Ames Co., Inc., Elkhart, Ind.). Reactions of 3+ and 4+ (>1 mg/ml) were considered as significant proteinuria. Hematocrit values were determined with a standard capillary tube technique. Hemoglobin determinations were done on 20-LI samples of blood using the cyan-hematin method. Anti-erythrocyte antibodies were determined by a direct Coombs reaction. The erythrocytes of each mouse were washed twice in warm PBS without Ca`+ and Mg2' at pH 7.4 and diluted to a 16% suspension in the same buffer. A drop of this suspension was layered on a glass plate and mixed with a drop of a suitable dilution of rabbit anti-mouse Ig antibody. Cells from 10-months-old NZB mice and from young NMRI mice served as positive and negative controls. Determinations of p30 and gp69/71 were done by radioimmunoassay, using the interspecies determinants (11, 19). The reaction mixture for immunoprecipitation contained the following: 0.003 ml of normal rabbit serum, 1 to 2 ng of '25I-labeled proteins (3 x 104 to 6.5 x 104 cpm/ng), and 0.01 ml of diluted rabbit anti-feline virus (Theilen strain). The final volume was adjusted to 0.2 ml with TEN buffer [20 mM tris(hydroxymethyl)aminomethane-hydrochloride (pH 7.6), 1 mM ethylenediaminetetraacetic acid, 100 mM NaCl] containing 2 mg of crystalline bovine serum albumin per ml and Triton X-100 to a final concentration of 0.2% in the assay. The mixture was incubated for 4 h at 37°C, after which 0.03 ml of goat anti-rabbit IgG was added to precipitate the antigen-antibody complexes. The mixture was incubated for 1 additional h at 37°C and then overnight at 4°C. Cold TEN buffer (0.5 ml) was added, and the precipitate was collected by centrifugation at 4°C. The pellet was washed twice with 0.5 ml of cold TEN, and the '25I-labeled antigen present in the precipitate was measured in a gamma counter. The competition radioimmunoassay reaction mixture was the same except that the antiserum dilution added was that which precipitated 50% of the labeled antigen. Competing proteins (spleen extracts and serum) were diluted in TEN buffer containing 2 mg of bovine serum albumin per ml and Triton X-100 to a final concentration of 0.2%. Anti-double-stranded DNA (dsDNA) antibodies were detected by immunofluorescence on Crithidiae luciliae (1) (kit supplied by the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands). FITC-labeled goat anti-mouse Ig antibody was used. Antinuclear antibody was detected by indirect immunofluorescence on frozen sections (4 tm) of normal rat liver. The sections were incubated for 30 min at room temperature with the mouse sera (1/10 dilution in PBS), washed three times with Veronal buffer (pH
7.2), stained for 30 min with FITC-labeled goat antiserum to mouse Ig, washed, and examined with a fluorescence microscope equipped with a Ploem illumination system. Immune complex detection in kidneys. Fresh kidney fragments were snap-frozen in isopentane, precooled in liquid nitrogen. The specimens were kept at -70°C until examination. Unfixed cryostat sections (4 ,tm) were air-dried, incubated with the appropriate antiserum, and examined with a fluorescence microscope equipped with a Ploem illumination system. For detection of mouse Ig precipitates, the sections were incubated for 30 min with a suitable dilution of FITClabeled goat antiserum to mouse 1g. For detection of C:, deposits, an indirect technique was used. The sections were incubated for 30 min with a suitable dilution of rabbit antiserum to mouse BC/BA globulin (C,). FITC-labeled goat antiserum to rabbit Ig was then applied for 30 min. The specificity and appropriate dilutions were tested using kidney sections of normal NMRI mice and old NZB/NZW-F, hybrids as negative and positive controls.
RESULTS Failure of interferon to reduce murine leukemia virus p30 and gp69/71 levels in NZB spleens. In a preliminary experiment, the effect of interferon treatment on the levels of the major C-type viral protein p30 and of the viral envelope glycoprotein gp69/71 in serum and spleen was determined. Fifteen NZB mice (27 to 32 days old) were divided into three groups receiving either PBS, mock interferon, or interferofh (104." units in 0.1 ml, intraperitoneally, five times weekly) during 8 weeks. Radioimmunoassay results for p30 and gp69/71 are shown in Table 1. In control mice, the p30 levels in the spleen and gp69/71 levels in the serum were comparable to those reported for NZB mice in the literature (19, 23); gp69/71 levels in the spleen were about twice as high, which might be accounted for by a different method for preparation of the spleen extract. Treatment with mock interferon or interferon did not influence any of these levels. From these data it was concluded that exogenously administered interferon is unable to affect the production of the major protein of the endogenous NZB C-type virus, or at least that higher dosages would be needed. Therefore, the dose of interferon was increased at least 10-fold in further experiments, in which the effect on the progression of disease was evaluated. Accelerated progression of hemolytic anemia in interferon-treated NZB mice. In NZB mice the main component of the autoimmune disease is hemolytic anemia as manifested by a positive Coombs reaction. The following experiment was done to study the effect of interferon on the progression of the disease. Thirty
INTERFERON IN NZB MICE
VOL. 21, 1978
NZB mice (15 males and 15 females; 29 to 36 days old) were randomly divided in three groups of 10. They were given PBS, mock interferon, or interferon (105'3 units in 0.1 ml, intraperitoneally, three times weekly) during 37 weeks. Table 2 shows spleen weights and incidences of antierythrocyte, antinuclear, and anti-dsDNA antibodies. The spleens of the female mice were consistently larger than those of the males. Both in males and in females the mean spleen weights of interferon-treated mice were higher than those of mice given PBS or mock interferon. However, all interferon-treated mice had also antierythrocyte antibodies, while more than 50% of the mice in both control groups were negative in this respect. Therefore, it seemed reasonable to conclude that, under the conditions of our experiment, interferon accelerated, rather than inhibited, the progression of NZB disease. The incidence of antinuclear and anti-dsDNA antibody was low in all groups and seemed not to be affected by the treatment. TABLE 1. Effect of interferon on p30 and gp69/71 levels in spleen and serum of NZB mice p30 proTreatment"
No: miceof 4"
PBS
gp69/71 protein
t Spleen
Spleen
Serum
(ng/mg)
(ng/mg)
(jig/ml)
(35.08)'
1,752.5 (442.66)
6.42 (1.94)
237.75
Mock interferon
5
291.00 (19.66)
1,947.2 (540.29)
7.20 (1.28)
Interferon
5
289.6 (39.16)
1,659.2
6.324
(607.86) (2.20) "Groups of five NZB mice were injected intraperitoneally with 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.
927
Accelerated progression of renal disease in interferon-treated NZB/NZW-F1 mice. To examine the effect of exogenous interferon on the development of renal autoimmune disease, 30 female NZB/NZW-F, hybrid mice (28 to 34 days old) were divided in three groups and were given either PBS, mock interferon, or interferon (10';" units per 0.1 ml, intraperitoneally, three times weekly) for 33 weeks. Blood and urine samples were taken after 9, 13, 17, 22, and 30 weeks and at the end of the experiment. Proteinuria, hemoglobin, hematocrit, and antibodies against erythrocytes, dsDNA, and nuclear DNA were determined. The kidneys were examined for presence of immunoprecipitates. Body weights and terminal data for urine and blood are summarized in Table 3. In each group the animals gained body weight till the end of the experiment. The interferontreated group was slightly behind on the two control groups. Extensive edema, which might explain the gain in body weight, was not found at autopsy. The spleen weights of interferontreated mice were not increased as seen in NZB mice. The mean values of hematocrit and hemoglobin were slightly but not significantly lower in interferon-treated mice. At the time of autopsy 1 of 10 PBS-treated mice, 2 of 10 mock interferon-treated mice, and 4 of 9 interferontreated mice had hematocrit values of s40%. Antierythrocyte antibodies were not detected except in terminal serum samples. Positive Coombs reactions occurred in two of nine interferon-treated mice and were completely absent in the two control groups. Taken together, these figures suggest an accelerated progression of anemia in the interferon-treated group. Antinuclear and anti-dsDNA antibodies were only determined in the terminal serum samples. Positive reactions were randomly distributed over the three groups.
TABLE 2. Effect of interferon on hemolytic anemia in NZB mice No. of animals with:
Mice Treatment"
Sexatme" Sex
Mean spleen wt"
No(mg) No.
Anti-erythro-
cyte antibody'
Antinuclear antibody
Anti-dsDNA antibody
1 2 4 6 144 (20.64) 3 250 (52.68) 0 2 4 83 (18.87) 4 Mock interferon Female 5 333 (79.70) 1 0 6 9 Male 230 (60.26) Interferon 3 Female 963 (452.6) "Groups of 10 NZB mice were injected intraperitoneally with PBS, mock interferon, or interferon (10" units in 0.1 ml) three times weekly during 37 weeks. One animal of each group died during the experiment, due to unknown 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 groups is significant at the P < 0.02 level (X2 test with Yates correction). PBS
Male Female Male
928
INFECT. IMMUN.
HEREMANS ET AL.
TABLE 3. Effect of interferon on the disease parameters of N7,B/NZW-F, hybrid mice Treatment"
No. of
mmice
PBS
5 mo
10
Mock interferon Interferon
Body wt. () at age: ; mo mo
7.5
8.5 mo
Spleen wt (mg)
% Erythrocytes
Hemoglobin
(g per l(X) ml)
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)
93
(8.119)
44.26 (0.9)
(0.19)
10
27.55
30.25
31.25
(0.639)
(0.484)
(0.716)
31.55 (0.651)
27.11
28.28 (0.434)
30.55 (0.733)
30.44
127
40.13
(0.757)
(5.094)
(1.66)
9'
(0.455)
15.11
14.08 (0.51)
No. of animals with: Treatment"
of No. mice
PositivereCoombs action
ANF'
Anti-DNA
Proteinuria -1 mg/ml
>1 mg/ml
Renal fluorescence Mild
Severe
10 0 6 PBS 5 8 2 8 2 0 10 Mock interferon 5 4 9 1 9 1 9' 2 7 Interferon 3 5 4 5 4 " Groups of female NZB/NZW-F, mice were injected intraperitoneally with PBS, mock interferon, or interferon (10" units in 0.1 ml) three times weekly during 33 weeks. All figures, except body weight, refer to terminal 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+) first appeared in week 17 of the experiment. It appeared later (week 30) in the interferon-treated mice. However, 4 of 9 animals in this group had severe proteinuria, against 1 of 10 and 2 of 10 in the two control groups. This slight difference prompted us to terminate the experiment to evaluate the kidneys immunologically. Fluorescence, revealing antibodies to mouse Ig and C:I, was demonstrated in the glomeruli of all mice in each group. It was graded in two patterns: (i) "mild" renal fluorescence with focal and lumpy deposits of Ig and C: preponderantly in the mesangial spaces of the glomeruli and, in a few instances, sparsely along some capillary basement membranes (Fig. 1A), and (ii) "severe" renal fluorescence with coarse deposits of Ig and C: 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 had severe fluorescence. In contrast, four of nine interferon-treated mice had severe fluorescence. From these data it is clear that the interferon treatment certainly had no beneficial effect on the development of the NZB/NZW autoimmune disease. On the contrary, interferon-treated animals appeared to have slightly higher incidence rates of proteinuria, anemia, and severe renal fluorescence. Table 4 shows the correlation in the occurrence of the different disease parameters that
were measured. When all mice were considered as one population, renal fluorescence, proteinuria, and low hematocrit and hemoglobin levels occurred in association with each other. Antinuclear and anti-dsDNA antibody seemed to occur independently from these parameters.
DISCUSSION Long-term treatment of the NZB with moderate doses of interferon depressed neither the spleen levels of p30 and gp69/71 nor the serum levels of gp69/71. Although the same doses of interferon were found to provide partial protection against exogenous infection with Mengo virus (our unpublished data), the failure of interferon to depress p30 and gp69/71 levels could be anticipated. Indeed, studies with chronically infected cell cultures have shown that the interferon-mediated inhibition of C-type virus replication is not accompanied by a depression in the synthesis of the major viral proteins (5, 17). Accordingly, it could also be predicted that interferon would not prevent NZB disease. However, it was unexpected that long-term treatment with high doses of interferon accelerated the development of disease symptoms in NZB and NZB/NZW-F, mice. The mechanism of this accelerated development can at present only be speculated upon. In fact, the effect may not even be due to interferon itself, but rather to impurities in the preparation. With each injection of interferon, the mice also received small amounts
VOL. 21, 1978
INTERFERON IN NZB MICE
929
FIG. 1. Fluorescent photomicrographs of renal glomeruli from 8.5-month-old NZB/NZW-F, mice treated with 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. Ig is deposited in the mesangium and along capillary wells. Comparable results occurred with antiserum to mouse C.. TABLE 4. Association of different NZB/NZW-F, disease parameters,with the extent of renal
fluorescence Renal fluorescence
Parameters Total Proteinuria (>1 mg/
Significance" Severe 7"'
Mild 22
0 7 P < 0.(01 ml) 6 Low hematocrit 2 P < 0.001 (G40%i) 7 Low hemoglobin (614 0 P < 0.001 mg/ml) 6 Antinuclear antibody 12 0.05 < P < 0.1 Anti-dsDNA anti2 10 0.75 < P < 0.8 body "Statistical significance of association with severe renal fluorescence by X2 test with Yates correction. " Number of mice in pooled groups (experiment shown in
Table 3).
of foreign antigens, such as proteins from bovine serum or residual Newcastle disease virus components. The effect of these antigens on the development of disease symptoms was only partially controlled by the inclusion of a group treated with a mock interferon preparation: this preparation did not contain Newcastle disease virus. The accelerated development of disease in interferon-treated mice has some parallels in the literature. Experimental infection with several viruses was shown to result in earlier development of nephritis in NZB mice (14, 21). The administration of a synthetic double-stranded RNA, polyinosinic acid-polycytidylic acid, and Tilorone, two potent interferon inducers, also
caused earlier disease symptoms in NZB mice (3, 18, 22). In contrast to these reports, a favorable effect was observed using natural doublestranded RNA (16). Recently, it has been reported that interferon treatment of suckling mice can lead to a progressive lethal glomerulonephritis in adult mice (6). Anti-interferon antibodies were shown to protect mice against chronic disease caused by lymphocytic choriomeningitis virus (15). Thus interferon, or molecules that are produced in association with interferon, might provoke rather than inhibit some of the symptoms of virus disease. A similar suggestion has come Lrom the observation that patients treated with interferon develop malaise, fever, and transitory lymphopenia and that intracutaneous injection of interferon elicits an inflammatory response
(4). ACKNOWLEDGMENTS This work was supported by a grant from the Belgian A.S.L.K. Cancer Research Foundation. The study on viral proteins was done during the tenure of an American Cancer Society-Eleanor Roosevelt-International Cancer Fellowship awarded by the International Union against cancer to A. C. on leave from the Institute of Pathological Anatomy, University of Padou, Italy. We are indebted to B. Van Damme and E. Meulepas for helpful discussions. Purified Rauscher murine leukemia virus p30 and gp69/71 and rabbit anti-feline leukemia virus sera were kindly provided by M. Strand, Albert Einstein College of Medicine, New York. Anti-dsDNA determinations were done by E. Stevens (Division of Immunology, A.Z. Pellenberg, Leuven). The technical assistance of Chris Neuckermans-Dillen, Francine Cornette, Lieve Godefridus, R. Conings, R. Wijnants, and P. Moerkerk, and the editorial help of Janine Putzeys, are appreciated.
930
HEREMANS ET AL. LITERATURE CITED
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