W F1 mice with NZW splenic T cells or with serum of mice experiencing the graft-versus-host reaction: Suppression of ongoing anti-double-stranded (ds) DNA antibody formation and improvement of renal function

CLINICAL

:MMUNOLOCY

AND

IMMUNOPATHOLOGY

32, 359-361 (1984)

Treatment

of NZB/W F, Mice with NZW Splenic T Cells or wit of Mice Experiencing the Graft-versus-Host Reaction: pression of Ongoing Anti-Double-Stranded (ds) DNA body Formation and Improvement of Renal Function

HIRCXAZLJ OKUDAIRA,*T~, L%MXRA GGHDA,*

EIJI TERADA,” KYOKO FUKUDA,*

TQSH~O ITO,* OGITA,?

TATSUJI NOMURA,” KOICHIRO KUDO,? TADAATSU AND TERUMASA

MIYAMOTO~

“Laboratory of Immunology, Central Institute for Experimental Animals. 1430 Nogawa, Miyamae, Kawasaki 213, and fDepartment of Internal Medicine and Physical Therapy, University of Tokyo, 7-3-l Hongo, Bunkyo-ku, Tokyo 113, Japan Seven-month-old NZBiW F, mice were inoculated with 5 x lo7 NZW spleen cells. The mice so treated showed a clear fall of anti-double-stranded (ds) DNA antibody titers accompanied by a decrease in blood urea nitrogen (BUN) concentration. Mice so treated showed markedly increased survival times (more than 17 months) compared with untreated controis (mean ? SD 8.38 + 0.75 months). A cell fractionation study suggested that T cells among NZW spleen cells play a major role. Recipients of the unfractionated or the T-cell fraction of NZW spleen cells had histologically less glomernlonephritis than untreated control NZBiW F, mice. NZW B-cell transfer to NZBiW F, mice had no effect. Sernm obtained from NZBiW F, mice receiving NZW spleen cells 10 days previously (graft-vs-host (GVHR) serum) was injected into R-month-old NZBiW F, mice. A clear fall in anti-ds DNA antibody titers and inhibition of age-associated sharp increases in BUN were observed, The mean life span of these mice (10.50 + 0.58 months) was significantly longer than that of untreated controls (8.69 I 0.38 months). GVHR serum bad the ability to suppress anti-ds DNA antibody formation by NZB/W F, spleen cells in vitro. Allogeneic GVHR sernm prepared in C57B116 x DBAR (BDF,) mice by transferring C57Bii6 spIeen ceIIs was also effective in suppressing anti-ds DNA antibody formation in vitro.

INTRODUCTION

It is well known that the F, hybrid of NZB X NZW mice (NZB/W F,) develop anti-double-stranded (ds) DNA antibody and a fatal immune complex glomerulonephritis remarkably similar to human systemic lupus erythematosus (SLE) (1, 2). For control of SLE and other autoimmune diseases, the regulation of autoantibody formation is apparently critical. In general, F, hybrid mice receiving par-ental strain lymphoid cells develop a graft-versus-host reaction (GVHR). The immunosuppressive effect of GVHR on the induction of the primary antibody response has been reported by several authors (3,4), but the conclusion was not directly applicable to the established anti-ds DNA antibody response in NZB/W IF, mice since the cellular mechanisms involved in the induction of primary antibody responses and in maintaining of the established antibody responses are quite different (5, 6). The effect of GVHR induction on autoantibody formation in the mouse was investigated and reported from several laboratories, but the 359 0090-1229184 $1.50 Copyright Q 1984 by Academic Press. Inc. All rights of reproduction in any form reserved

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conclusions were not consistent and it seemed to be dependent on experimental conditions (7-9). The present study showed suppression of anti-ds DNA antibody formation, improvement of renal function, and marked prolongation of the life span of NZB/W F, mice that were inoculated with NZW spleen cells. The effective cell population was suggested to be T cells. It was interesting to note that such activities could be transferred to untreated NZB/W F, mice by the serum of NZB/W F, mice administered NZW spleen cells. An in vitro assay system for the immunosuppressive activity of GVHR sera was devised. MATERIALS

AND METHODS

Mice. Female NZB, NZW, NZB/W F,, C57Bl16, and BDF, mice maintained at the Central Institute for Experimental Animals, Kawasaki, Japan, were used. Mice were housed in filter-capped cages. Cellfractionation. Depletion of T cells in the spleen was carried out using antimouse Thy 1.2 (Miles-Yeda Ltd., Rehovot, Israel) and guinea pig complement as previously described (10). The number of T cells contaminated in the fraction was 2 to 3% of the total cells, as determined by a cytotoxicity test. To obtain the T-cell-enriched fraction, Ig-bearing cells were removed from spleen cells using anti-mouse Ig-coated dishes following the method described by Mage et aE. (11). The proportion of T cells in the T-cell-enriched fraction was 70 to 75%. Ig-bearing cells remaining in the fraction were 1 to 2% of the total cells, as determined by immunofluorescence. Anti-ds DNA antibody assay. Details of solid-phase radioimmunoassay for antids DNA antibody are described elsewhere (12). Briefly, dilutions of mouse sera or culture supernatants were added to ds DNA-coated tubes and uncoated control tubes. After incubation at 37°C for 120 min, the solution was aspirated and the tubes were washed three times with excess washing buffer. 1251-anti-mouse immunoglobulin solution was added to each tube, and the tubes were incubated overnight at room temperature and then washed three times with washing buffer. The radioactivities retained on the tubes were counted in an automatic gamma counter. Pooled serum obtained from 10 female NZBiW F, mice, aged 9 to 10 months, was arbitrarily assigned an anti-ds DNA antibody concentration of 10,000 units/ml. Standard curves were prepared using this serum to determine the amount of anti-ds DNA antibody in each serum or culture supernatant. Suppression of anti-ds DNA antibody formation in vitro. Spleen cells were obtained from a NZB/W F, mouse showing clear anti-ds DNA antibody formation and suspended at a concentration of 5 x 106/ml in a culture medium consisting of RPM1 1640 supplemented with 10% fetal calf serum (FCS), 5 x 1O-5 N 2mercaptoethanol, 5 mM glutamine, 100 units penicillin, and 100 pg/ml streptomycin. Equal volumes of the cell suspension and various preparations of mouse sera, diluted 1:2 with culture medium or a control culture medium, were mixed, and 0.2 ml of the cell suspensions was placed in the wells of Falcon Microtest II plates (Falcon Plastics). Cell cultures were performed in a humidified incubator with a gas flow of 5% CO, and 95% air. Culture supernatants were recovered on Day 8 and were subjected to anti-ds DNA antibody measurement.

GRAFT-VERSUS-HOST

REACTION

IN NZB/W

MICE

361

UN measurement. BUN measurement was performed for rough estimation of the glomerular filtration function by the urease method using BUN500 Sinotest (Sinotest Laboratory, Tokyo, Japan). The BUN of 6-month-old nonautoimmune BDF, mice was 16.7 ?I 4.9 mg/dl. Statistical analysis. Statistical comparisons were performed by the Student t test. RESULTS

&‘j%ct of parental strain spleen cell transfer to NZBIW F, mice. Groups of four ‘I-month-old NZB/W F, mice were inoculated with 5 x lo7 spleen cells obtained from NZW or NZB mice. Four untreated NZB/W F, mice of the same age served as controls. Part of the experimental results are shown in Fig. 1. Anti-ds DNA antibody titers rapidly increased in the control group, generally associated with age. In the group receiving NZW spleen cells, a clear decrease of anti-ds DNA antibody titers was observed. The antibody titers reached the minimum levels in months 8 to 9 and gradual recovery took place. The BUN progressively increased in the control group until the mice died, while mice inoculated with NZW spleen ceils showed a marked fall in BUN half a month after cell transfer and stayed suppressed for more than 3 months. Control mice died before 10 months of age (mean 2 SD 8.38 r4: 0.75 months), but NZB/W F, mice administered NZW spleen cells survived for more than 17 months. In the NZB/W F, mice that were inoculated with NZB spleen cells the anti-ds 2OOr

H

Transfer

NZW

(B)

o--e 0-0

Cel I Transfer

NZW Cel I Transfer Control

Transfer

FIG. 1. Effect of NZW spleen cell transfer to NZB/W F, mice on anti-ds DNA antibody formation (A) and BUN concentration (B). 5 x 10’ NZW spleen cells were transferred to 7-month-old NZB/W F, mice (GVHR group O-0). Untreated NZB/W F, mice of the same age served as control (O---O). Mice which died before next bleeding are marked (t). A clear fall in anti-ds DNA antibody titers and BUN concentrations was observed in the GVHR group. Mice in the GVHR group lived more than 17 months; while control mice died before 10 months (8.38 2 0.75 months).

OKUDAIRA

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ET AL.

DNA antibody titer fell from 4175 -t 1782 to 350 + 58 units/ml, and BUN levels dropped from 20.0 +- 7.4 to 15.2 t 7.5 mg/dl by half a month after cell transfer. However, all mice died before 10 months (8.75 + 0.50 months) and prolongation of survival was not observed. Spleen cell fractionation study and histological analysis. In a subsequent experiment NZW spleen cells were fractionated into T-cell-enriched (T-cell) and Tcell-depleted (B-cell) fractions by the methods described under Materials and Methods. Groups of four 7.5-month-old NZB/W F, mice were inoculated with 5 x lo7 unfractionated cells, or the T- or B-cell fractions of NZW spleen cells. Untreated NZB/W F, mice of the same age served as control. Mice were bled just before and 14 and 28 days after the cell transfer and the blood was assayed for anti-ds DNA antibody and BUN. The results of the experiment are shown in Table 1. Control and B-cell transferred mice showed a clear increase in anti-ds DNA antibody concentration, except for those which wasted to death before next bleeding. A remarkable fall of anti-ds DNA antibody formation was observed in mice which received unfractionated cells or the T-cell fraction of NZW spleen cells. BUN concentrations on Day 28 stayed low or improved in these animals compared to the concentrations on Day 0. Mice in the control and B-cell transferred groups showed increases in or persistent high levels of BUN concentration. Kidneys of mice in critical condition were collected before death for histological analysis. Remaining mice were sacrificed at 10 months of age. Eight mice that TABLE TRANSFER

OF NZW

NZW spleen cell fraction transferred”

1

SPLENIC T CELLS SUPPRESSES Aim-ds AMELIORATES RENAL FUNCTION IN

Anti-ds DNA antibody (units/ml) Day 0

Day 14

Day 28

Day 14

Day 28

47 61 55 47

58 111 59 36

46 48 35b 32

74 61 68 61

49 86 61 43

43 48 436 39

13,000 20,000

61 52 68 47

59c 59 67 61

68 67 62

20,000 10,400 20,000 -

58 80 74 41

105 130 80 2ooc

114 152 80 -

650 500 5506 750

650 200 500b 700

T cell

6400 6000 4400 3100

700 450 650b

1,100 200”

1,000

6300 5700 4900 4200

3,800’ 8,800 13,000 6,300

6350 4800 4700 4600

8,200 5,500 6,300 650c

AND

Day 0

6950 6600 5000 3100

None

FORMATION

BUN (mg/dl)

Unfractionated

B cell

DNA ANTIBODY NZB/W MICE

1,200

1,600

18,800

’ 5 X 10’ fractionated NZW spleen cells were transferred to 7..5-month-old NZB/W F, mice. b The mean titers were significantly lower than those of no-cell transferred control mice (P < 0.01). c Wasted and died before next bleeding.

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were inoculated with unfractionated cells or the T-cell fraction of NZW spleen cells were alive, while only two mice in the B-cell transferred group and one mouse in the no-cell transferred control group were alive at 10 months. In the kidneys of mice inoculated with the B-cell fraction of NZW spleen cells or not treated, pathological changes were observed more or less in all glomeruli. Glomerular tufts were lobulated and adhered to Bowman’s capsule. Mesangial matrix was increased and the basement membrane of capillaries was thickened which sometimes showed wire-loop-like lesions. Hyaline thrombi were occasionally observed. Cellular proliferative change was not conspicuous except epithelial cell proliferation of Bowman’s capsule. Local tubular atrophy, containing an eosinopbilic substance in it, was also remarkable. No tubular necrosis was found (Fig. 2). Gn the other hand, in the kidneys of NZB/W F, mice inoculated with unfractionated cells or the T-cell fraction of NZW spleen cells, only segmental proliferative changes of the glomerular tufts were occasionally seen. Pathological change of the tubuli was not found (Fig. 3). Effect of GVHR serum administered to NZBIW F, mice. Eight-month-old NZBIW F, mice received five daily injections of 0.25 ml GVHR serum which was prepared in NZB/W F, mice by transferring (iv) 5 x IO’ NZW spleen cells 1 days before sacrifice. Control NZB/W F, mice received no treatment. Experimental results are shown in Fig. 4. All of the four NZB/W F, mice in the control group died before 10 months of age (8.69 ? 0.38 months), while all mice administered GVHR serum survived more than 10 months (10.50 t 0.58 months). The

FIG. 2. Glomerulonephritis of a control, untreated NZB/W F, mouse. Proliferation of mesangial cells accompanied by a thickening of capillary walls is shown. The capillary lumens became narrow. Marked dilatation of urinary tubuli was observed. PAS x 200.

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FIG. 3. Low grade glomerulonephritis of a kidney of a NZB/W F, mouse which received 5 x lo7 NZW spleen T cells. No remarkable change was observed except a slight hypercellularity in the glomerular matrix. PAS x 200.

difference between the mean life spans of the two groups of mice was statistically significant (P < 0.01). A clear decrease in anti-ds DNA antibody titers was seen in mice receiving GVHR serum. The fall was most obvious 1 week after serum transfer. The anti-ds DNA antibody titer before GVHR serum administration was 9500 + 1842 units/ml, but fell to 5031 t 1351 units/ml half a month after GVHR serum infusion. The titers remained suppressed for a month and gradually increased thereafter. The age-associated increase of BUN levels was suppressed in the group of mice treated with GVHR serum. In vitro assay of immunosuppressive activity of GVHR sera. A IO-month-old NZB/W F, mouse showing a high titer (10,000 units/ml) of anti-ds DNA antibody in the serum was sacrificed to obtain spleen cells for in vitro anti-ds DNA antibody formation. Syngeneic GVHR sera were prepared in NZBiW F, mice by transferring 5 x lo7 NZW or NZB spleen cells. The allogeneic GVHR serum was taken from BDF, mice administered 5 x lo7 C57B116 spleen cells. GVHR sera were collected 10 days after cell transfer. A NZBiW F, spleen cell suspension was mixed with syngeneic or allogeneic GVHR sera or a culture medium and incubated for 8 days. After culture, supernatants were collected and assayed for antids DNA antibody. Control supernatants, in which NZB/W F, cells were cultured without mouse serum, contained 195 units/ml of anti-ds DNA antibody. Coexistence of normal syngeneic (NZB/W F,) or allogeneic (BDF,) mouse serum in the culture exerted no clear influence on anti-ds DNA antibody formation, while GVHR sera induced significant suppression. The syngeneic GVHR serum pre-

GRAFT-VERSUS-HOST 2Q,OOO

REACTION

(A)

200

IN NZBiW

365

MICE

(B) Q+

I

OLA Gt;iR Serum

H 0-a

GVHR Control

Age

(Months)

Serum

i.v.

I

9

Ib

(1

OL;(

4 G%R Serum

i.v.

,b Age (Months)

111

ix.

FIG. 4. Effect of iv injection of GVHR serum in NZB/W F, mice on anti-ds DNA antibody formation (A) and BUN concentration (B). GVHR serum was formed in NZB/W F, mice by transferring 5 x IO’ NZW spleen cells 10 days previously. Eight-month-old NZB/W F, mice received five daily injections of 0.25 ml of GVHR serum (GVHR serum group; O-O). Untreated NZB/W F, mice of the same age served as control (O---O). Mice which died before next bleeding are marked (i). A clear fall in anti-ds DNA antibody titers was observed in the GVHR serum group. The age-associated rapid increase in BUN concentration was not seen in the GVHR serum group. Mice in the GVHR serum group lived significantly longer than those in the control group (P < 0.01).

pared by transferring NZW or NZB spleen cells showed 46 and 39% suppression, respectively, while allogeneic GVHR serum showed 65% suppression. DISCUSSION

The data in the present study clearly showed that NZB/W F, mice inoculated with NZW spleen cells develop a clear fall of anti-ds DNA antibody formation accompanied by a decrease in BUN concentration (Fig. 1). At the beginning of the experiment, we found no correlation between anti-ds DNA antibody titers and BUN levels in the NZB/W F, mouse, but the fall in anti-ds DNA antibody titers apparently correlates with the decrease in BUN levels; thus the formation of large amounts of,anti-ds DNA antibody may not be a sufficient condition but may be a necessary condition for the development of glomerulonephritis. A similar observation was made by Lehman et al., who reported that the incidence of increased proteinuria and mortality was reduced in NZB/W F, mice which had been inoculated with NZW spleen cells at 5-6 months of age before establishment of anti-DNA antibody formation (7). They measured anti-native DNA antibody at approximately 8 months of age using a modified Farr technique to find that percentage binding of native DNA by the control and experimental groups were 9.97 and 5.15, respectively. The palliative effect of the NZW spleen cell-induced GVHR on glomerulonephritis which Lehman et al. and we observed is at variance

366

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with the increased autoantibody formation by NZB-induced GVHR in NZBiW F, mice described by Goldblum et al. (8). The difference in results can not necessarily be attributed to the two kinds of parental cells used, since in our study NZB/W F, mice given with NZB spleen cells also showed suppression of anti-ds DNA antibody formation and a fall in BUN level. The difference may originate from the protocol followed by Goldblum et aZ., which employed weekly injections of NZB cells to induce GVHR (8). Elven et al. (9) reported that a GVHR can produce anti-ds DNA antibody in nonautoimmune strains of mice. The authors postulated abnormal cooperation between alloreactive donor helper T cells and recipient B cells. It is probable that a subset of T cells playing a major role in different experimental systems is qualitatively different. Marked increase of survival times of NZB/W F, mice transferred with NZW spleen cells (more than 17 months) compared with untreated controls (mean rf: SD 8.38 t 0.75 months) prompted us for further analysis. A cell fractionation study suggested that NZW splenic T cells play a major role in the prolongation of the life span of NZB/W F, mice. Recipients of unfractionated cells or the Tcell fraction of NZW spleen cells showed a remarkable fall of anti-ds DNA antibody formation, while BUN concentrations stayed low or improved after cell transfer (Table 1). NZBiW F, mice given unfractionated cells or the T-cell fraction of NZW spleen cells showed histologically less glomerulonephritis than untreated control and B-cell transferred animals (Figs. 2 and 3). Despite the possible beneficial effect, GVHR is essentially a dangerous procedure which is sometimes fatal to animals. Therefore, an attempt was made to examine whether serum taken from mice experiencing GVHR could mimic GVHR itself after transfer to untreated NZB/W F, mice. It was interesting to note that administration of GVHR serum partially but clearly substituted for a GVHR in suppressing anti-ds DNA antibody formation and BUN accumulation (Fig. 4). Since the autoimmune disease processes of NZB/W F, mice are quite heterogeneous, and anti-ds DNA antibody titer, BUN concentration, and survival time of each mouse vary considerably, establishment of more quantitative assay systems for the immunosuppressive activity of GVHR serum is required. Recently, we have succeeded in establishing microculture systems of NZBiW F, spleen cells to form anti-ds DNA antibody in vitro (12), and an attempt is being made to assay the immunosuppressive activity of GVHR serum using the system. The immunosuppressive activity has been successfully measured in vitro and it has shown that not only syngeneic GVHR sera but also allogeneic BDF, GVHR serum could suppress anti-ds DNA antibody formation by NZB/W F, spleen cells. The immunosuppressive factor(s) appeared to work across the histocompatibility barrier. It was interesting to note that nonautoimmune BDF, GVHR serum had the greatest suppressive activity. Autoantibody formation may be suppressed in nonautoimmune strains of mice by abundant immunosuppressive factor(s) in the serum. The mechanism by which NZW spleen cells transferred to NZB/W F, mice exert immunosuppressive activity is unclear, and the possibility that the direct attack of NZW cells on NZBIW F, antibody-forming cells plays some role remains at present, although it is more probable that immunosuppressive factor(s) elab-

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orated by NZW splenic T cells depress the function of NZB/W F, antibodyforming cells. The lack of a graft-versus-host reaction against NZW cells in NZBlW F, mice might favor the survival and functioning of NZW spleen cells for a long period of time. The finding that GVHR serum was effective in prolonging longevity of &month-old NZB/W F, mice in which anti-ds DNA antibody formation and azotemia had been established suggested the possibility that the ameliorating factor(s) could be used in clinical therapy in the future. Characterization and mass production of the immunosuppressive factor(s) are now under investigation. REFERENCES 1. Howie, J. B., and Helyer, B. J., In “Advances in Immunology” (F. .I. Dixon and H. 6. Kunkel, Eds.), Vol. 9, p. 215, Academic Press, New York, 1968. 2. Lambert, P. H., and Dixon, F. J., J. Exp. Med. 127, 507, 1968. 3. Branden R. V., Transplantation 7, 484, 1969. 4. Davis, W. E., Jr., Cole, L. J., and Schaffer, W. T., Transplantation 9, 529, 1970. 5. Okudaira, H., and Ishizaka, K., Cell. Zmmuno/. 58, 188, 1981. 6. Qkudaira, H., Terada, E., Ghoda, A., Ogita, T., Miyamoto, T., and Nomura, T., Znt. Arch. Allergy Appl. Zmmunol. 70, 376, 1983. 7. Lehman, D. H.; Wilson, C. B., and Dixon, F. J., Clin. Exp. Zmmunol. 25, 297, 1976. 8. Goldblum, R., Pillarisety, R., Dauphinee, M. J., and Talal, N., C/in. Exp. Zmmunol. 19, 377, 1975. 9. Elven, E. H., Veen, F. M., Rolink, A. G., Issa, P., Duin, T. M., and Gleichmann, E., J. Zmmunol. 127, 2435, 1981. 10. Okudaira, H., and Ishizaka, K., J. Zmmunol. 114, 615, 1975. 11. Mage, M. G., McHugh, L. L., and Rothstein, T. L., J. Zmmunol. Methods 15, 47, 1977. 12. Okudaira, H., Terada, E., Ogita, T., Aotsuka, S., and Yokohari, K., J. Zmmunol. Methods 41, 201, 1981. Received January 23, 1984; accepted with revisions May 14, 1984.