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Effect of altered lymphocyte function on immunologic disorders in nzbnzw mice.

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65
EFFECT OF ALTERED LYMPHOCYTE
FUNCTION ON IMMUNOLOGIC
DISORDERS IN NZB/NZW MICE
I. FAVORABLE RESPONSE TO L-ASPARAGINASE
JAGDISH MEHTA, LINDA L. KNOTTS, and BEVRA H. HAHN
NZB/NZW F, female mice were treated with the
immunosuppressive enzyme L-asparaginase ( Asnase) for
up to 32 weeks. Asnase diminished circulating anti-DNA
and antinuclear antibodies, diminished deposition of yglobulins in kidneys, significantly delayed the onset of
proteinuria, and reduced deaths from nephritis. These effects were associated with reduction of cellular IgM antibody synthesis to both T-dependent and T-independent
antigens, but the graft-versus-host reaction was not affected. After several weeks of therapy, antibodies against
Asnase appeared in the circulation, the effect od antibody
synthesis was lost, ANA and anti-DNA appeared, followed by proteinuria and deaths from nephritis. Therefore
Asnase proved to be an effective therapy in NZB/NZW
mice, but its usefulness was limited by the appearance of
inactivating antibodies.
NZB/NZW F, mice develop lethal immune complex glomerulonephritis accompanied by antibodies to
From the Department of Medicine, Washington University
School of Medicine, St. Louis, Missouri.
Supported by USPHS Research Grant R01 AM 17469 and
Training Grant TO1 AM05548.
Jagdish Mehta, Ph.D.: Fellow, Rheumatology Division, Department of Medicine; Linda L. Knotts, B.Sc.: Washington University
School of Medicine; Bevra H. Hahn, M.D.: Assistant Professor of
Medicine, Washington University School of Medicine.
Address reprint requests to Jagdish Mehta, Ph.D., Rheumatology Division, Washington University School of Medicine, 660
South Euclid, St. Louis, Missouri 631 10
Submitted for publication April 19, 1976; accepted September 17, 1976.
Arthritis and Rheumatism, Vol. 20, No. 1 (January-February 1977)
nucleoprotein, DNA, and RNA. Glomerular immune
deposits contain antibodies to DNA and t o murine type
C viruses (1,2). Antibodies directed against erythrocyte
surfaces also occur in a significant proportion of these
mice (3,4). Numerous studies (5-7) have shown that the
circulating antibody responses of New Zealand mice to
numerous antigens are increased compared to the responses of healthy mouse strains.
Decreased cellular immune responses accompany
the increased humoral responses in these mice. Evidence
for reduced TGcell function as the mice age includes
reduced responses of spleen cells to mitogens, decreased
ability to reject allografts, relative resistance to induction of tolerance, and the development of antilymphocyte antibodies with predilection for T cells (8-14).
One of the likely mechanisms for production of
undesirable antibodies in NZB/NZW mice is overfunction of B cells; this abnormal actitdty may well
result from loss of T-cell suppressor functions. The evidence for this concept has been reviewed by Steinberg
(15).
Efforts to ameliorate the immune nephritis of
New Zealand mice could be directed toward improvement of T-cell function or suppression of B cells. LAsparaginase (Asnase) is an immunosuppressive enzyme
that reportedly reduces both T and B cell functions.
It is thought to act by “starving” rapidly dividing
cells (especially of the lymphocyte series) for asparagine.
In mice, Asnase has been reported to diminish the following T-dependent functions: cellular antibody syn-
66
thesis t o sheep erythrocytes ( S R B C ) , graft-versus-host
reactions, allograft rejection, and irl vitro responses t o
PHA (16-18). I t h a s been reported t o diminish t h e following B-cell functions: in vitro synthesis of IgG by r a t
spleen cells and transfer of sensitivity t o SRBC by bone
m a r r o w cells (19,20).
Hence an a t t e m p t was made to suppress undesirable antibodies by treating young female NZB/NZW
mice with Asnase for 32 weeks. T h e y were compared to
untreated female littermates.
MATERIALS AND METHODS
Animals
NZB/NZW F, mice were bred from stock colonies of
NZB/BI and NZW mice maintained by the Rheumatology
Division at Washington University. All mice used in the study
were female.
Study Design
Four-week-old NZB/NZW F, females were randomized into two groups of littermates, each containing 60 mice.
The first group received L-asparaginase (Merck, Sharp and
Dohme, West Point, PA), 10 international units ( I U ) per day,
5 days a week, inoculated intraperitoneally. This therapy was
continued for 32 weeks, after which mice were sacrificed. The
second group was not treated.
After 6 and 20 weeks of therapy, groups of treated and
control mice were sacrificed to obtain spleen cells for measurement of plaque-forming cells (PFC) to SRBC and to pneumococcal polysaccharide (SSSIII), and for graft-versus-host reactions. Kidneys were snap-frozen and later y-globulins were
eluted from pools of renal tissue and quantitated. Fifteen to 20
mice in each group were permitted to live for the entire 32
weeks to provide survival data. All mice were studied at 4week intervals for proteinuria (measured by AlbustixH) and
blood urea nitrogen levels (estimated with AzostixH). Serums
obtained at the same 4-week intervals were studied for antinuclear antibodies (ANA), antibodies to nDNA, and antibodies
to Asnase.
Test Procedures
Measurements of spleen PFC after immunization with
SRBC were performed by using a modified Jerne assay, as
described by Jacobs and colleagues (21). Mice were inoculated
with 3 X 10' SRBC intravenously and spleens were assayed for
PFC on day 4, which was the day of maximal response. SSSIII
was kindly provided by Dr. Benjamin Prescott of the Microbiology Division of the National Institutes of Health, Bethesda. Immunization with SSSIll and measurement of direct
PFC to that antigen on day 5 (maximal response) were performed by the method of Baker and colleagues (22); SSSIIl
was coupled to carrier erythrocytes in chromium chloride.
Indirect PFCs were not measured in any of the systems. For all
PFC tests, assays were performed in triplicate; background
MEHTA ET AL
PFC from cells incubated with uncoated SRBC were subttacted from final results.
Graft-versus-host reactions were measured by the
modification of the Simonsen assay described by Cantor and
associates (23). Cells (2 X 10') teased from NZB/NZW
spleens and washed in Hank's balanced salt solution were
inoculated intraperitoneally into newborn C57B I /6 Cum
mice. The recipients were sacrificed 9 days later, and the ratio
of spleen weight to body weight was determined for each
mouse. This ratio was compared to the ratio in an untreated
littermate. and the final result was expressed as the "GVH
index."
Studies of renal tissue were performed by eluting and
quantitating y-globulins. After 6 and 20 weeks of treatment,
kidneys from all mice in each group were pooled and homogenized. Globulins were eluted by the acid elution method of
Lambert and Dixon ( I ) and quantitated by the gel diffusion
method of Mancini el a1 (24).
Antinuclear antibodies (ANA) were measured by indirect immunofluorescence using undiluted mouse serums and
mouse kidney as substrate, as previously described (25). Antibodies to nDNA were measured by a modified Farr assay,
using tritiated KB DNA (Electronucleonics, Bethesda, M D )
as antigen, as described previously (26).
Antibodies against L-asparaginase were detected by the
ability of test serums to interfere with degradation of asparagine. The coupled enzyme system described by Cooney et al
(27), which measures the continuous hydrolysis of L-asparagine, was used for the Asnase assay. Briefly, to 1.9 ml 0. I M
Tris buffer (pH 8) were added 50 p 1 (5 p M ) asparagine, 50 pl
( 1 p M ) a-ketoglutarate, 50 pl (0.5 p M ) NADH, 50 pl ( I IU)
glutamic oxaloacetic transaminase (all from Sigma Chemical
Co, St. Louis, MO). After temperature equilibrium a t 37OC,
the reaction was started by the addition of either 50p1(0.2 1U)
I.-asbaraginase o r 50 pl of reaction mixture containing Asnase
and booled serums from treated or untreated mice of different
ages. Appropriate controls were included in all assays. The
change in optical density at 340 nm was measured in a Beckman model Acta M IV spectrophotometer. One unit of Lasparaginase was defined as that amount of enzyme which
hydrolyzes I pM of L-asparagine per minute at 37°C. Results
were expressed as units of Asnase detectable in the assay and
converted to percentage of inhibition by test serums.
Antibodies to L-asparaginase were not detectable by
gel diffusion methods. However the inhibition of L-asparaginase activity by test serum was thought to be due to antibodies
against L-asparaginase. Serums lost their ability t o inhibit
Asnase activity either after ammonium sulfate precipitation of
y-globulins or after treatment with rabbit antimouse y-globulin.
RESULTS
Effects on Immune Responses
T r e a t m e n t with Asnase resulted in reduction of
spleen cell synthesis of IgM antibodies t o SRBC, a Tdependent antigen, as shown in Table 1. T h i s effect w a s
d e m o n s t r a b l e after 6 weeks of treatment, before antiAsnase antibodies appeared a t 8 weeks, but n o t after 16
ALTERED LYMPHOCYTE FUNCTION
Table 1. Effect of L-Asparaginase Therapy on Spleen Cell Synthesis
of IgM Antibodies to Sheep Erythrocytes
After 6 Weeks Treatment
No Treatment
Asparaginase
After 20 Weeks Treatment
No Treatment
Asparaginase
~
PFC/Spleen ( X 10')
4.8
3.0
4.3
2.0
4.2
1.5
3.3
0.9
3.1
0.6
3.0
0.6
5.1
4.9
4.8
4.6
4.3
3.2
P
< 0.05*
2.2
0.8
0.5
0.5
-
P
> 0.10*
Antibodies were significantly reduced after 6 weeks of treatment but
not after 30.
* P determined by the Mann-Whitney U test (43) and by pair
analysis (44).
to 20 weeks. Reduction of this antibody response can be
attributed to diminished T- or B-cell function, or both.
As Table 2 shows, spleen cell synthesis of IgM
antibodies to SSSIII, a T-independent antigen, was also
reduced after 6 weeks of therapy but not after 20. SupTable 2. Effect of L-Asparaginase Therapy on Spleen Cell Synthesis
of IgM Antibodies to Pneumococcal Polysaccharide SSSIII.
a T-Independent Antigen
After 6 Weeks Treatment
No Treatment
Asparaginase
After 20 Weeks Treatment
No Treatment
Asparaginase
PFC/Spleen ( X l O ' )
2.9
0.5
2.9
0.4
2.6
0.4
2.3
0.3
1.6
0.3
1.6
1.2
I .o
2.9
2.6
2.6
2.5
2.4
2.2
2.1
I .9
P
< 0.05*
0.5
0.3
0.3
0.2
2.1
2.6
2.3
2.2
1.9
I .6
pression of these antibodies was also demonstrable at 9
weeks, but not at 16 (data not shown). Reduction of this
antibody response implies suppression of B-cell function.
Data from the graft-versus-host reactions are
shown in Table 3 . Spleen cells from untreated controls
and from mice treated for 4 weeks with Asnase produced similar GVH indices. In the authors' experience,
spleen cells from female NZB/NZW mice at 8 weeks of
age have a reduced capability to induce G V H when
compared to cells from healthy mouse strains; therefore
G V H indices do not often reach 2.0. There was no
reduction of G V H in the Asnase group after 4 or 20
weeks of therapy. Therefore Asnase-induced reduction
of T-cell function could not be demonstrated.
Effects on A N A Formation
As Figure 1 illustrates, therapy with Asnase delayed the appearance of antinuclear antibodies (ANA)
for 12 weeks. Thereafter, the proportion of mice with
positive tests increased rapidly. After 12 weeks of treatment, 14 of 29 untreated mice were ANA positive, compared to 4 of 30 treated mice ( P < 0.01 by xz analysis).
Therefore the effect of Asnase on antibody synthesis
resulted in suppression of these undesirable antibodies
for several weeks.
Similarly, antibodies to nDNA were temporarily
suppressed by Asnase treatment, as shown in Figure 2.
Many pretreatment serums from mice randomized to
receive Asnase contained anti-nDNA; the mean level of
DNA binding was 14.4% f 2.0 SEM. In contrast, mean
binding of DNA by pretreatment serums from untreated
controls was 6.9% f I .O SEM-significantly lower than
Table 3. Effect of L-Asparaginase Therapy
on Graft- Versus-Host Reactions
1 .o
P
> 0.1
PFC/I06 ( X 10')
2.1
0.3
2.5
0.3
1.8
0.2
I .7
0.2
I .6
0.2
1.4
0.9
2.9
2.8
67
After 4 Weeks Treatment
0.5
0.2
0.2
0.1
No Treatment
Asparaginase
P
> 0.1
Antibodies were significadtly reduced after 6 weeks, but not after 20.
* P determined by pair analysis (44).
t P determined by the Mann-Whitney U test (43).
No Treatment
Asparaginase
G VH Indices
1.93
I .03
I .84
0.1 I
1.60
0.64
1.30
0.55
1.03
2.06
1.61
I .39
I .38
I .08
0.1
P < 0.025t
P < 0.05*
After 20 Weeks Treatment
P
> 0.1*
1.27
I .09
1.05
0.19
0.14
0.61
0.9 < P > 0.05
See text for details of this method. Asnase treatment did not reduce
the ability of spleen cells to induce GVH after 4 or 20 weeks of
treatment.
* P determined by the Mann-Whitney U test (43).
68
MEHTA ET AL
r
v)
I-
v)
80Lu
-
delayed for 8 weeks by Asnase therapy. By x2 analysis,
the proportions of mice with 2+ proteinuria or greater
were significantly lower in the Asnase group after 16,20,
and 24 weeks of therapy. Deaths from nephritis (mice
dying with 2+ proteinuria or greater and blood urea
nitrogen >20 mg% were classified as renal deaths) began
approximately 8 weeks after the appearance of proteinuria in both groups and were therefore delayed for 8
weeks by Asnase therapy. Deposition of mouse y-globulins in renal tissues was diminished in the Asnase group
after 6 and 12 weeks of treatment, as shown in Table 4.
A
No R,
L
t
v)
9 60I
t
Antibodies Against Asparaginase
WEEKS OF
TREATMENT
4
8
12
ANTIBODIES
TO ASNASE
0
t
ttt
16
20
I
nt
Fig 1. Effect of Asnase treatment on the development of antinuclear
antibodies ( A N A ) . The percentages of mice with positive A N A are
shown. At I2 weeks the proportion of mice with positive A N A was
significantly lower in the Asnase-treated group. After 12 weeks antibodies to Asnase (indicated by arrows) developed. Thereafter, A NA
increased and was similar to the control group. See Table 5 for antibodies to Asnase. Asterisk: P < 0.01 by ,y2 analysis.
Serum pools from each group began to inhibit the
activity of Asparaginase after 8 weeks of treatment with
Asnase, as shown in Table 5. At subsequent 4-week
intervals, serum inhibition increased and approached
100% after 12 weeks of treatment. The inhibiting activity
resided entirely in ammonium sulfate precipitable proteins. Serums lost their inactivating properties after
treatment with rabbit anti-mouse y-globulins. It seems
likely that the inhibition was caused by antibodies. The
inactivation of Asnase by these antibodies probably ac-
t--. N O R x
in the Asnase group (P< 0.01 by student’s t test). AntiDNA measurements were made in all serums at the
conclusion of the study, so the difference in pretreatment values had not been anticipated. The high
antibody levels in the Asnase group at the beginning of
treatment were fortuitous, because Asnase was capable
of reducing that antibody titer. From 0 to 12 weeks of
treatment, the mean binding dropped in the Asnase
group from 14.4% f 2.0 SEM t o 8.4% f 0.8 SEM (P <
0.01 by student’s t test). In the control group DNA
binding rose from 6.9% f 1.0 to 15.8% f 3.8 (P <
0.025). After 12 weeks the percentage of DNA bound by
serums from treated mice was significantly lower than
the percentage bound by control serum (P < 0.01).
Thereafter, anti-DNA antibody levels rose rapidly in
both groups.
Effects on Clinical Disease
As expected, the suppression of circulating ANA
and anti-DNA was associated with suppression of nephritis, as shown in Figure 3. The onset of proteinuria was
o--o
Asnase
;
0
6
12
16
3-
WEEKS OF TREATMENT
Fig 2. Eject of Asnase treatment on anti-DNA antibodies. Mean percentages of radioactive nDNA bound by serums from each group are
shown. Anti-DNA activity was decreased during the first I2 weeks of
therapy; binding was significantly lower in the Asnase group at 12 weeks.
Thereaper, effectiveness of Asnase was lost, probably because of the
development of anti-Asnase antibodies. See Table 5 for antibodies to
Asnase. Asterisk: P < 0.01 by Mann- Whitney U test.
ALTERED LYMPHOCYTE FUNCTION
F
3
z
69
~~
DEATHS FROM NEPHRITIS
PROTE INURI A
I
c
.
No R,
8ot
80
c"
k
u
z
w
? 60
60
2
6
0
40
n
2
n
I-
w
(L
Z
2ol
4
w
20
12
20
28
WEEKS
g
a
OF TREATMENT
Fig 3. Effects of Asnase therapy on development ofproteinuria and deathsfrom nephritis. The percentages of
mice in each group that developed greater than 2 + proteinuria and died from renal disease are shown.
Proteinuria was signifcantly lower in the Asnase group after 16.20, and 24 weeks of treatment. Deaths from
nephritis were delayed for 8 weeks after the appearance of proteinuria. Asterisks: signif cant difference by x1
analysis.
counts for the emergence of ANA in the treated group
after 12 weeks, followed by proteinuria 8 weeks later.
DISCUSSION
There has been much interest in the development
of potential new therapies for chronic immune complex
diseases such as systemic lupus erythematosus (SLE).
The use of corticosteroids and of cytotoxic drugs is
common in these disorders but associated with undesirable and sometimes fatal toxicities.
The abundant evidence for defective cellular immune responses (8-15,28) in mice and humans with
SLE, with subsequent hyperactivity of the humoral immune response (5-7,15), suggests that therapies deTable 4. Effect of L-Asparaginase Therapy on Renal
Deposits of y-Globulin
After 6 Weeks Treatment
After 12 Weeks Treatment
N o Treatment
Asparaginase
No Treatment
Asparaginase
10.6*
5.6
31.2
9.1
* All measurements expressed as pg of Ig/g of kidney tissue.
signed to specifically manipulate T- or B-cell function
might alter the course of these diseases. For example,
further reduction of T-cell function by administration of
antithymocyte globulin (ATG) has resulted in marked
acceleration of nephritis (29,30). This effect was associated with decreased T suppressor effect on the antiSSSIII antibody response, increased circulating antibodies t o DNA, and increased deposition of mouse
y-globulin and anti-DNA in renal tissue. These data
Table 5. Inhibition of Asparaginase Activity by Serum from
Asparaginase-Treated Mice
Weeks of
Treatment
0
4
8
12
20
Detectable Units
of Asnase in
100 p1 Reaction
Mixture*
0.2
0.2
0. I66
0.0053
0.0026
Percent
Inhibition
by Serums
0
0
17
97.4
98.8
Serums from control mice at the same treatment intervals showed
no inhibition of asparaginase activity.
* 0.2 IU added in each test.
70
suggested that loss of T-cell suppressor function permits
increased B-cell production of undesirable antibodies.
Other evidence for this hypothesis has been reviewed by
Steinberg ( 1 5).
Attempts have been made to ameliorate the
nephritis of NZ mice by improving T-cell function.
Transfer of syngeneic lymphocytes from young mice to
older mice who have developed T-cell defects resulted in
reduction of autoantibodies and prolongation of life
(3 1,32). In contrast, administration of thymosin (thymic
hormone) to the mice failed to alter their clinical disease
(33), although there is in vitro evidence that thymosin
can restore properties of defective T cells in NZB and
NZB/NZW mice (34-36).
Another approach to therapy would be reduction
of B-cell function and subsequent antibody production.
The authors’ experiments with L-asparaginase suggest
that this approach is feasible and does ameliorate the
disease process in murine lupus. Several investigators
have reported that i n vivo administration of Asnase
results in diminished T-cell function, decreased delayed
hypersensitivity responses in humans (37), prolonged
allograft survival in mice (38), and diminished in vitro
responses to PHA, specific antigens, and allogeneic lymphocytes (19). In addition, B-cell functions are also
clearly inhibited, as indicated by suppression of IgG
synthesis by rat spleen cells incubated in vitro with
Asnase ( I 9), by reduced in vitro transformation of lymphocytes in the presence of lipopolysaccharide (39), and
by the observations of Friedman that the ability of
murine marrow cells (but not thymic cells) to respond to
immunization with SRBC was inhibited by Asnase
treatment (20).
The present experiments showed suppression of
IgM antibody formation to SSSIII, a T-independent
antigen, as well as antibodies to a T-dependent antigen
(SRBC) and antibodies to nuclear antigens. These results strongly suggest the inhibition of B-cell antibody
synthesis. The lack of effect on GVH suggests that T-cell
function was relatively unaffected. However NZB/
NZW mice at 8 weeks of age already have T-cell defects, and the authors may have been unable to further diminish GVH. The decreased GVH response in 24week-old NZB/NZW mice in these experiments differs
from the report of Gerber et al (40), who showed increased GVH response at 24 weeks. This discrepancy
might be explained by the fact that the current study
used a different mouse strain as recipient and a different
dose of spleen cells. The possibility of Asnase altering
balance between helper and suppressor T cells cannot be
excluded.
MEHTA ET AL
Suppression of antibody synthesis to DNA and
other nuclear antigens was associated with prolonged
survivals, delayed proteinuria, and diminished deposition of y-globulins in renal tissue. These effects were
temporary; ANA began to appear after 12 weeks of
treatment.
Studies of Asnase therapy of malignant tumors
have revealed two problems that limit the therapeutic
value of this agent. First, repeated administration of
Asnase to mice is followed by induction of increased
asparagine synthetase activity in spleen cells (39). Second, circulating antibodies against Asnase appear and
apparently are capable of partially inactivating the agent
(4 1.42). In the present experiments inactivation of Asnase by the globulin fraction appeared in pooled serums
from treated mice after 8 weeks of treatment, and inhibition was approximately 100%after 12 weeks. Anti-DNA
and ANA appeared in the circulation of treated mice
after 12 weeks, followed 8 weeks later by proteinuria
and then death from nephritis. If mice could be rendered
tolerant to Asnase before regular administration of this
enzyme is begun, perhaps it would be effective for longer
periods of time.
Could this therapy, which is dramatically although temporarily beneficial in murine lupus, be applied to human SLE? It is always hazardous to extrapolate data from mouse to man. However Asnase has been
used as a therapeutic agent in the management of human acute leukemias. In that situation it has been associated with significant toxicity, including fatty change of
liver, hypoalbuminemia, anasarca, pancreatitis, diabetes
mellitus, and elevated blood ammonia levels. These side
effects are at least partly dependent on dose and duration of therapy (37). Although the toxicities differ from
those of cytotoxic agents, there is obviously some overlap with the undesirable side effects of corticosteroids.
The authors are not advocating the use of Asnase in
human S L E at this time, although there might be a role
for carefully supervised, short-term therapy added to
standard regimens in patients hospitalized with lifethreatening disease.
Whether or not Asnase proves useful in the management of SLE, it is clear that relatively selective suppression of B-cell function is beneficial in the animal
model of this disease and may be applicable to other
immune complex disorders.
ACKNOWLEDGMENTS
The authors thank Dr. John Rogers of the Hematology Division for allowing us to use his Beckman Acta MIV
ALTERED LYMPHOCYTE FUNCTION
spectrophotometer, and Ms. Evelyn Stenseth for secretarial
assistance.
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