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Study of the multiple factors in the pathogenesis of autoimmunity in new zealand mice.

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S190
STUDY OF THE MULTIPLE FACTORS
IN THE PATHOGENESIS OF
AUTOIMMUNITY IN
NEW ZEALAND MICE
ALFRED D. STEINBERG, L. W. KLASSEN, E. S. RAVECHE, N. L. GERBER, J. L. REINERTSEN,
R. S. KRAKAUER, D. F. RANNEY, M. E. GERSHWIN, G. W. WILLIAMS, K. KOVACS,
and J. P. REEVES
Autoimmune New Zealand mice provide animal
models of human systemic lupus erythematosus. Investigations suggest that the disease is multifactorial in origin;
genetic, viral, biochemical, and hormonal factors are all
capable of modifying the expression of autoimmunity.
However, the immune system is the common pathway of
disease expression. Early abnormal functioning of the
immune system involves excessive effector function and
inadequate suppressor function. Since New Zealand mice
act as if they are excessively stimulated, the defect in
suppressor function may be relative rather than absolute.
Thus, excessive B-cell stimulation cannot be controlled
by available regulatory processes.
Thymic developmental abnormalities point to an
early primary defect in the generation of regulatory T
cells. As NZB and NZB/NZW mice pass from neonatal
to young adult life, they lose suppressor cell function,
probably from the loss of one of at least two cell types
From the Arthritis and Rheumatism Branch, National Institute of Arthritis, Metabolism. and Digestive Diseases, National Instittttes of Health.
Alfred D. Steinberg, M.D.: Senior Investigator, Arthritis and
Rheumatism Branch, National Institute of Arthritis, Metabolism, and
Digestive Diseases, National Institutes of Health, Bethesda, Maryland; L. W. Klassen, M.D.: Research Associate, NIAMDD, NIH; E.
S. Raveche, Ph.D.: Research Associate, NIAMDD, NIH; N. L. Gerber, M.D.: Clinical Associate, NIAMDD, NIH; R. S. Krakauer, M.D.:
Department of Immunology, Research Division, Cleveland Foundation, Cleveland, Ohio; D. F. Ranney, M.D.: Department of Microbiology/Immunology, Northwestern University Medical School, Chicago; M. E. Gershwin, M.D.: Clinical Associate, NIAMDD, NIH;
G. W. Williams, M.D., Ph.D.: Clinical Associate, NIAMDD, NIH;
K. Kovacs: Clinical Associate, NICHD, NIH; J. P. Reeves, M.S.:
Chemist, NIAMDD, NIH.
Address reprint requests to Alfred D. Steinberg, M.D.,
National Institute of Arthritis, Metabolism, and Digestive Diseases,
National Institutes of Health, Building 10, Room 8D19, Bethesda,
Maryland 20014.
Arthritis and Rheumatism, Vol. 21, No. 5 Supplement (June 1978)
that interact to produce suppression. The lost cell appears
to be an immature or precursor cell. Anti-T cell antibodies
and immune complexes may accelerate the loss of suppressor cells and thereby lead to more rapid depletion
of the suppressor cell precursors.
Beneficial therapeutic efforts include administration of suppressor cells or their products, steroid
hormones-glucocorticoids and androgens-immunosuppressive drugs, antiinflammatory agents, and antiviral
drugs.
The autoimmune diseases of NZB and NZB X
NZW F, mice have been intensively investigated with
regard to both pathogenesis and therapy. The immunologic features of these NZ mice closely resemble those of
patients with SLE. Therefore, it has been hoped that
better understanding of the mice will improve our ability
to deal with the human condition.
The etiology of disease in NZB mice and their
hybrids has been elusive; however, a number of pathogenetic mechanisms have been implicated in disease production (1-5). Perhaps the most widely accepted is immune complex renal disease. A variety of immune
complexes probably contribute to the production of glomerulonephritis. The best demonstrated are antinuclear
antibodies and antibodies to murine leukemia virus and
the several antigens with which those antibody populations combine. One of the most puzzling aspects of
New Zealand mouse disease has been why the right
types and quantities of antibodies are produced to promote the formation of this immune complex disease.
Additional uncertainties have revolved around the formation of antibodies with particular specificities, especially those which recognize DNA and mouse erythrocytes.
AUTOIMMUNITY IN NZ MICE
Clues concerning the production of these abnormal antibodies have been sought primarily among the
immunologic derangements found in these mice. These
include abnormal histological development of the
thymus; premature loss of thymic hormone; abnormal
thymic and T cell immune regulatory function; selective
B cell hyperresponsiveness; macrophage dysfunction.
The excessive production of autoantibodies could be
due to excessive B cell function as a primary disorder or
to impaired T cell regulation of B cell function, o r both.
Evidence for both abnormalities is accumulating. New
Zealand mice act as if they are stimulated with an immune enhancer (adjuvant) and their immune suppressor
functions are inadequate to control the resultant B cell
hyperfunctioning. The precise relationships between abnormal thymic development, abnormal T cell function,
thymic hormone levels, and autoantibody production
remain to be determined.
NZB mice and their hybrids carry one o r more
types of murine leukemia viruses which have been implicated as the cause of the disorder. The viral hypothesis
with regard to etiology of the lupus-like disease of New
Zealand mice is particularly attractive. First of all, it
provides a single causative agent which is easily understandable. Secondly, there is the precedent of other immune complex “autoimmune” diseases being caused by
a virus (for example Aleutian mink disease and chronic
lymphocyte choriomeningitis virus infection). Thirdly, it
provides an agent against which therapeutic efforts
might be directed. In support of the viral hypothesis,
there is clear-cut evidence that NZB mice have a greater
viral load than most other mice and that immune complexes of viral antigens and antibodies to those antigens
lodge in the kidneys. However, most of the antibody in
the kidney is not directed toward viral antigens. In addition, breeding studies do not suggest a relationship between amount of murine leukemia virus and autoimmune features. Finally, viral infection has not been
shown to be responsible for other abnormalities found
in New Zealand mice (the attempts have not been complete so that this is still an open question). Thus, the
viral hypothesis remains unproved. It is likely, however,
that viral infection accounts for some of the abnormalities of New Zealand mice-even if only secondarily.
Thus the autoimmune diseases of NZB and NZB
X NZW F, mice appear to be complex with regard to
pathogenetic mechanisms. Although viral and genetic
factors have been implicated as causes of the disorder,
such factors appear to operate through the immune
system. As a result, the studies in this laboratory have
been directed toward derangements in the immune system of t h e New Zealand mouse in a search for greater
S191
understanding of the pathogenesis of their autoimmune
manifestations. Special attention was given to the time
before development of overt illness in order to avoid
emphasis of immunologic abnormalities secondary to
the disease process.
MATERIALS AND METHODS
Animals. Mice were obtained from the Rodent and
Rabbit Production Unit at the National Institutes of Health.
Special matings were carried out in our laboratory. All mice
had continual access to food and water. Studies using very
young animals (less than 1 week old) were performed on the
offspring of mice placed in the laboratory 5 or more days prior
to delivery. Lethally irradiated and reconstituted mice received
pH 2-2.4 drinking water for several weeks after irradiation.
Castration. Testes were removed through a single
scrota1 incision. Controls had the scrotum opened and the
testes exposed. Incisions were closed with 5-0 silk sutures.
Oophorectomies were performed through bilateral flank incisions. The skin incision was made over the kidney and a
small incision was made in the peritoneum over the lower pole
of the kidney. The ovary was exposed and removed without
ligation of vessels. The skin was closed with 5-0 silk sutures.
Sutures were not required for bleeding or peritoneal closure.
Sex Hormone Treatment. Initial treatment was by injection. Testosterone propionate in peanut oil was given in
0.05 ml subcutaneously to both males and females from 3
weeks of age in a dosage schedule of 25 pg daily. Estradiol
valerate in sesame oil was given in 0.05 ml subcutaneoulsy
to males and females from 3 weeks of age in a dosage schedule
of 5 pg daily. In the past two years treatment has consisted
of implantation of capsules containing either testosterone
(4 mg) or estradiol (2 mg) under the skin of the back of the
neck.
NTA Assay. Assay for naturally occurring thymocytotoxic antibody (NTA) was performed as previously described (6). In brief, washed C57B1/6 thymocytes were labeled
with “Cr and incubated with serial dilutions of sera. Fresh
frozen rabbit serum was used as a source of complement. The
last twofold dilution giving 50% kill was taken as the titer of
the serum. A titer of >1:4 was considered positive. Complement alone or with negative DBA/2 serum gave < 15% kill
at a dilution of 1 : 1 .
Neonatal Thymectomy. NZB/NZW mice, less than 18
hours of age, were thymectomized under a dissecting microscope using a suction catheter and light ether anesthesia. Approximately 80% of the mice survived. Completeness of thymectomy was demonstrated by postmortem examination that
included serial histologic sections of the mediastinum.
Thymus Grafts. The thymus was carefully removed
from donor animals in order to minimize the chance of transferring mediastinal lymph node tissue. The thymus was placed
into medium 199 (Microbiological Associates, Bethesda,
Maryland) and divided into two lobes. Each recipient received
one lobe implanted subcutaneously near the right axilla
through a sterile 13-gauge trocar.
Antibodies to DNA. Antibodies to DNA were assayed
by a modified Farr technique in which antibody bound to “C
labeled DNA was precipitated by ammonium sulfate (7,8).
Graft Versus Host Disease (GVH). Swiss mice less
s192
than 18 hours of age were injected with spleen cells in a volume
of 0.05 ml medium (9). A 30 gauge needle was inserted through
the thigh into the peritoneal cavity for this procedure. The
spleen weights and body weight of recipients were determined
9 days later. The spleen index was the ratio of spleen weight
experimental to body weight experimental divided by spleen
weight control to body weight control.
Unit Gravity Sedimentation. A single cell suspension of
NZB/NZW thymocytes was separated over a 5-30% linear
gradient of fetal calf serum by sedimentation in a chamber
without centrifugation as described previously (10).
Skin Graft Placement. Full thickness skin grafts were
prepared from male C57B1/6 donors approximately 6 weeks
of age. Recipients’ backs were shaved and the graft site was
prepared under pentobarbital anesthesia by removing an eliptical piece of skin approximately 5 cm long and 3 cm wide. The
graft was sewn into place with interrupted 6-0 silk stutures.
Grafts were inspected daily and their health (percentage of area
viable) was recorded without the examiner’s knowledge of
treatment. The graft was considered to be rejected when 110%
of the graft was viable.
Anti-Thy 1.2 Serum. Anti-Thy 1.2 serum was prepared
by injecting CIH mice with AKR thymocytes, as has been
previously described (6).One day prior to skin grafting, 0.5 ml
of appropriately diluted anti-Thy 1.2 was injected intraperitoneally into skin graft recipients. Controls received normal mouse serum.
In Vitro IgM Synthesis and Its Suppression. BALB/c
spleen cells were used as the source of cells for IgM synthesis.
Additional cells were added in order to assess their ability to
regulate the magnitude of the synthesis (1 1). These regulating
cells were obtained from either BALB/c or NZB/W mice and
were precultured with Concanavalin A, 2 pg/ml. One million
cells from each source were co-cultured and the amount of
IgM synthesis was compared to that obtained by co-culturing
the indicator BALB/c spleen cells with cells that were not pretreated with Con A (12). The sources of the Con A activated
cells included saline-treated or untreated BALB/c mice; and
NZB/W mice treated with saline, NMS, anti-Thy 1.2, NTA,
or thymocyte-absorbed NTA.
Studies of Coombs Test in NZB Mice. NZB mice 1 2
weeks of age were thymectomized and a single cell suspension
prepared. These cells were injected into recipient NZB mice
every 2 weeks starting at 4 weeks of age. Some of the recipients
received thymocytes from mice that had been treated 30 hours
prior to thymectomy with methyl prednisolone, 100 mg/kg.
Other recipients received thymocytes that were x-irradiated in
vitro with 2500 R or that were frozen and thawed. Mice were
bled approximately once a month and the direct Coombs test
assessing RBC-bound antibody was performed (13). The degree of glomerular disease, extent of reticulum cell hyperplasia,
and number of periarteriolar (T-lymphocytes) cells in the
spleen were graded without knowledge of treatment (14).
Study of Supernates from Spleen Cells. Single cell suspensions were prepared from NZB/W, BALB/c, and C57B1/6
mice and cultured at 3 X lV/ml in RPMI 1640 containing 2%
fetal calf serum for 60 hours at 37OC in a humidified air 5%
C 02 environment (15). The supernatant fractions were obtained by centrifugation and assayed. Additional cultures were
prepared in which spleen cells were cultured for either less than
5 minutes or for 40 hours with Concanavalin A, 2 pg/ml, and
the cultures were harvested (11). Con A was removed by
STEINBERG ET AL
passage of the supernate three times over pretreated G-75
Sephadex. Supernates from the various cultures were separated with regard to size by filtration through Amicon Filters.
Supernates from the 40-hour Concancavalin A cultures are abbreviated CONS to indicate a lack of prejudice
with regard to the active principle(s).The term CONS control
is applied to the less than 5-minute Con A culture supernates.
Various supernates were added to test lymphocyte suspensions (3 X 1V cells/ml) for 1 hour, at which time tritiated
thymidine was added. The cultures were allowed to go an
additional 4 hours and the degree of incorporation of the label
into DNA was determined (15).
Study of the Immune Response to SRBC. Mice treated
with CONS or control CONS were immunized in vivo with 2
X 1P SRBC iv, or their spleen cells were cultured in vitro with
SRBC as described in greater detail elsewhere (16). The number of antibody forming cells was determined by the technique
of local hemolysis in agarose. IgG plaques were developed
with antimouse IgG sera.
RESULTS
Relationship Between Sex and Sex
Hormones, and the Development of
Autoimmunity
Female hybrid offspring of NZB mice tend t o
have more severe disease manifestations than d o their
male littermates. Early studies of this phenomenon were
inconclusive in assigning the difference to sex hormones
or to immune response type genes on the sex chromosomes. W e have reinvestigated this phenomenon and
conclude that sex hormones are important in explaining
male-female differences in NZB hybrids.
T h e first study involved castrating male and female N Z B X N Z W F, mice at 3 weeks of age. A portion
of each group was treated with androgens or estrogens
and their natural history was followed with regard to
development of anti-DNA antibodies, proteinuria, and
survival (Table I). Male NZB X NZW mice that were
castrated developed earlier onset of high titers of antiDNA antibodies and proteinuria and died much earlier
than did untreated controls. Estrogen treatment did not
significantly alter this effect. In contrast, androgen treatment prevented the accelerated autoimmunity. Females
that were castrated did not differ from untreated females
or from castrated females treated with estrogens. In
contrast, females treated with androgens lived much
longer than untreated controls and also delayed development of anti-DNA antibodies and proteinuria (Table
l).* I n contrast t o these results produced by sexual
* Similar results have recently been reported by Roubinian et
a/. in J Clin Invest 591066, 1977.
AUTOIMMUNITY IN NZ MICE
S193
Table 1. Role of Androgens in Modulating the Expression of Autoimmunity in N Z B X N Z W F, Mice
Studies at 7 Months of Age
Sex
Treatment*
Sham
Castrated
Castrated + estradiol
Castrated + testosterone
Sham
Castrated
Castrated + estradiol
Castrated + testosterone
Sham + testosterone
An ti-DNA?
Ocg/ml)
30
79
87
32
f
f
f
f
21
31$
30$
18
114
98
107
46
33
f
f
f
f
f
32
38
34
22$
17$
Median
Proteinuria
(mg/100 ml)
Median
Survival
(days)
502
3401I
366 I I
469
30 1
334
316
402 I I
458 I I
Twenty mice per group.
t Antibody to native DNA (not rendered completely double stranded) in pg of antibody per ml of serum,
mean f SEM.
$ Significantly different from controls, P < 0.01, Student’s r test.
8 Significantly different from controls, P < 0.05, test of medians.
I I Significantly different from controls, P < 0.01, Kolmogorov-Smirnoff test.
alteration a t 3 weeks of age, similar studies initiated at
10 weeks of age failed to convert a male pattern of
autoimmunity into a female pattern, and vice versa,
although minor changes in those directions were noted.*
Thus, there appears to be an important maturational
event that is sex hormone sensitive and that takes place
prior to sexual maturity. This event probably involves
T-cell maturation such that androgens favor suppressor
rather than helper T cells.
The second study, performed by Raveche, Klassen, and Steinberg (17), involved hybrids of NZB and
DBA/2 mice. In these hybrids the spontaneous production of naturally occurring thymocytotoxic antibody
(NTA) was measured at 1 year of age. Female mice
developed such antibodies, whereas the males did not
(Table 2). However, when the males were castrated they
developed NTA with the same incidence as their female
littermates. Castration did not affect NTA production in
females. Additional control males that were sham castrated or vasectomized were not significantly different
from unmanipulated males. In additional studies of
these hybrids of NZB and DBA/2 mice, performed by
Raveche, mice were immunized with =DNA-MBSA
and the antibody response was measured. Preliminary
results indicate that females respond with a significantly
However, Drs. Katalin Kovacs and Alfred Steinberg have
recently found that implants of capsules containing testosterone prolong life in intact female NZB/W mice when implanted after the onset
of antinuclear antibody formation, at 3.5 months of age (manuscript
in preparation).
higher antibody titer than males, but that castration of
the males leads t o a significant increase in titer which is
comparable to that of the females.
A final series of studies has been performed by
Madeleine Duvic, who as a student in our laboratory
tried t o treat female NZB X NZW mice with nafoxidine,
an agent causing a major decrease in expression of estrogen receptors on membranes. She found that such treatment led to a significant decrease in antibodies to DNA
and increase in survival (18).
We conclude from these studies that sex differences in NZB hybrids may be attributed in large measure to sex hormone effects. Androgens appear to be
protective with regard to rapid development of autoimmunity, whereas estrogens appear to play a lesser role in
promoting autoimmunity.
Studies of Suppressor Cells
For several years we have been studying the relationship between immune regulation and the development of autoimmunity. The first experiment demonstrating that New Zealand mice lose suppressor cells
with age was performed by Steinberg in collaboration
with Law and Tala1 in 1968. These were subsequently
published in 1970 (19). They found that neonatal thymectomy accelerated the development of autoimmunity.
This could be prevented by a 2-week-old syngeneic
thymus graft, but not by a 10-week-old syngeneic
thymus graft (Table 3). Thus, between 2 and 10 weeks of
STEINBERG ET AL
Table 2. Accelerated Autoantibody Production by Male NZB Hybrids
Table 3. Accelerated Production of Antibodies to DNA in NZBIW
Following Castration
Female Mice Following Neonatal Thymectomy'
~
~~~
~~~
Antibodies to ssDNA 14 Days after Immunization
Animal
Sex Treatment
~
NZB X DBA
F
F
M
M
None
Castration
None
Castration
14
19
18
18
Sham operation
Thymectomy
Thymectomy
Thymectomy
~~~~
29.9 & 4. I
22.6 f 2.7
11.4 f 1.4,
29.1 f 2.9
Mean % % of Mice
Number Cytotoxicity Positive
Studied
f SEM
(> 50%)
Animal
Sex Treatment
NZBXDBA
F
F
M
M
M
None
Castration
None
Vasectomy
Castration
44
22
27
28
36
42.2 f 3.9
48.4 f 5.3
22.5 f 3.0
23.3 f 5.2
42.5 f 3.9t
48
F
F
M
M
None
Castration
None
Castration
7
10
8
8
58.4 f 10.6
72.1 & 3.9
60.7 f 10.2
80.3 f 2.2
71
90
F
F
M
M
None
Castration
None
Castration
5
9
6
13
27.0 f 6.9
18.9 f 6.3
12.0f 3.6
22.1 f 5.2
DBA/2
P
* Significantly lower than
< 0.01.
25.7
42.8
21.4
41.0
* Suppression of the accelerated production by syngeneic thymic
grafts from 2-week-old, but not from 10-week-old donors.
Spontaneous NTA Production at One Year of Age
NZB
None
None
2-Week thymus
10-Week thymus
Treatment
Mean %
DNA Bound
f SEM
Number
Studied
Graft
Native DNA Bound at
4 Months of Age
(Farr Assay), B
55
0
0
53
88
100
0
0
0
0
the other three groups, Student's t test,
Significantly higher than the other two male groups, Student's
t test, P < 0.01, but not significantly different from the two female
groups, P > 0.30.
age, the NZB/W thymus lost its capacity to regulate
autoantibody production. Furthermore, thymectomy at
6 weeks of age led to no acceleration of disease. In fact,
NZB/W females thymectomized at 6 or 10 weeks of age
lived 2-3 months longer than sham thymectomized controls. Subsequently, these studies have been extended.
NZB mice lose suppressor function for control of antibody responses to certain thymic independent antigens
after 3-4 weeks of age (20,21). NZB/W mice were
shown to lose suppressor cells for regulation of a cellmediated immune function, graft-versus-host disease
(GVH) (22,23). Thus, thymocytes from 4-week-old
NZB/W mice could suppress the GVH disease induced
by 18-week-old NZB/W spleen cells in newborn allogeneic recipients (23).
Thymocytes from 8-week-old NZB/W mice were
less effective but still significantly suppressive. However,
by 12 weeks of age, the thymocytes were not significantly suppressive (Table 4). Furthermore, the thymocytes from I-month-old NZB/W mice could be separated by unit gravity sedimentation into several fractions (24). Gerber and Steinberg found that one fraction
was greatly enriched in cells with suppressive function in
comparison to two other fractions that were greatly
enriched in cells with helper functions (Table 5 ) . Thus,
young NZB/W thymuses contain both helper and suppressor cells (25). When the mice are between 4 and 12
weeks of age, the cells with suppressor function are lost,
leaving an excess of cells with helper function. Later in
life, helper cells are also lost.
Studies of suppressing of skin allograft rejection
by anti-theta serum performed in our laboratory have
extended earlier studies of Gelfand and Paul (24). The
work has been carried on primarily by Klassen (26). A
single injection of anti-Thy 1.2 (anti-theta) into a mouse
Table 4. GraJi Versus Host Disease (Measured by Degree of Relative
Splenomegaly) Induced in Newborn Allogeneic Recipients by
4.5-Month NZBl W Spleen Cells*
Number of Cells Added
1 X l(P thyrpocytes
1 X 10"thymocytes
1 X 10"thymocytes
I X 10"thymocytes
0.5 X lo*thymocytes
5 X IO'thymocytes
0.5 X IVspleen cells
1 X lo6spleen cells
1 X l(P spleen cells
Age of Donor
0
4 weeks
8 weeks
12 weeks
16 weeks
4 weeks
4 weeks
4 weeks
4 weeks
16 weeks
Spleen Index
1.75 f 0.04
1.29 f 0.097
1.34 f 0.107
1.58 f 0.10
1.71 f 0.16
1.45 f 0.06t
1.14 f 0.08t
1.53 f 0.10
1.30 f 0.097
1.88 f 0.12
* Suppression by thymocytes and spleen cells from NZB/W mice
of different ages.
t Significant suppression.
S195
AUTOIMMUNITY IN NZ MICE
Table 5. Separation of Thymocytes from 4- Week-Old NZBl W Mice
by Unit Gravity Sedimentation into Fractions with Either Helper
or Suppressor Function
Table 7. Transfer of Suppression by Spleen Cells from Mice Treated
with Anti-Thy 1.2
Donor of Spleen
Fraction from
4-Week NZB/W Thymus
(1 x 109
Spleen Index
4.5-month NZB/W
4.5-month NZB/W
4.5-month NZB/W
4.5-month NZB/W
0
Fraction 1
Fraction 2
Fraction 3
1.62
1.21*
1.77
1.65
12-month NZB/W
It-month NZB/W
12-month NZB/W
12-month NZB/W
0
Fraction I
Fraction 2
Fraction 3
1.22
Spleen Cell Source
(5
x 1P)
Recipient
1.11
1.40t
1.577
* Significant suppression.
t Significant augmentation.
leads to prolonged skin allograft survival which is transferable with spleen cells. Such anti-Thy 1.2-induced suppression was elicited in 5-week-old NZB/W mice, but
not in 17-week-old NZB/W mice (Table 6). Of particular interest, the transfer of spleen cells from a 5-week-old
anti-Thy 1.2-treated NZB/W to another 5-week-old
NZB/W mouse transferred the suppression. In contrast,
transfer into a 17-week-old NZB/W was ineffective
(Table 7). However, transfer of cells from a 17-week-old
pretreated with anti-Thy 1.2 into a 5-week-old recipient
led to suppression (Table 7).
These studies suggest that an NZB/W mouse at
17 weeks of age may have one of at least two cell types
or functions that participate in suppression-the one
that is activated by or interacts with the anti-Thy 1.2.
The 17-week-old mouse lacks the second cell type that is
present in the 5-week-old mouse-the cell that is actiTable 6 . Suppression of Primary Skin Allograji Rejection by
Pretreatment with Anti-Thy I .2*
Recipient?
BALB/c-5
BALB/c-5
BALB/c-l7
BALB/c--17
NZB/W-5
NZB/W-5
NZB/W-l7
NZB/W-l7
weeks
weeks
weeks
weeks
weeks
weeks
weeks
weeks
Treatment
Mean Graft Survival f SEM
NMS
Anti-Thy 1.2
NMS
Anti-Thy 1.2
NMS
Anti-Thy 1.2
NMS
Anti-Thy 1.2
10.4 f 0.8
14.5 f 0.7$
10.2 f 0.6
13.9 f 0.7$
9.4 f 0.7
13.2 f 0.8$
9.9 f 1.0
8.3 f 0.8
* Each recipient received 0.5 ml of serum 24 hours prior to placement
of a full thickness graft of male C57B1/6 skin.
t 10-12 mice per group.
$ Significantly different from NMS group, P < 0.05, Student’s t test.
NZB/W-I7
NZB/W--I7
NZB/W-5
NZB/W-5
BALB/c--17
weeks
weeks
weeks
weeks
weeks
Cells Pretreated
with Anti-Thy 1.2
Suppression of
Skin Allograft
Rejection
0
0
NZB/W-17 weeks
NZB/W--5 weeks
NZB/W--5 weeks
NZB/W-17 weeks
BALB/c-17 weeks
+
+
t
vated by or recruited by the first cell type. Thus the
defect in suppression as NZB/W mice age may be manifested by the loss of only one of the cell types that
participate in suppression. An alternative possibility is
that 17-week-old mice have cells producing factors that
suppress the suppressor cell, which mediates the second
phase of suppression. We shall test this possibility by
putting all relevant cell types from 5-week-old donors
into the 17-week-old recipients.
Studies of NTA
Klassen has conducted a series of studies in our
lab in collaboration with Krakauer (12) to determine the
potential pathogenic role in the acceleration of suppressor cell loss produced by naturally occurring thymocytotoxic antibody, NTA, described in New Zealand
mice by Shirai and Mellors (27). Accordingly, baby
NZB/W mice, 3 to 5 days of age, were injected with
NTA or normal mouse serum (NMS) daily. At 4.5
weeks of age, these mice were studied for suppressor
function. Spleen cells from the NTA-treated mice induced a significantly greater GVH response in newborn
Swiss mice (Table 8). Further, such spleen cells were
incapable of generating suppressor cells in response to
Concanavalin A stimulation (Table 9). Thus, NTA appears to accelerate suppressor cell loss.
Table 8. Graft Versus Host Disease Induced in Newborn AIIogeneic
(Swiss) Recipients by 5 X I@ Spleen Cells from
1 Month-Old NZB/ W Mice*
Treatment
NMS
NTA
Number
Spleen Index
23
1.92
2.857
13
* All mice treated from 4 days of life with either naturally occurring
thymocytotoxic antibody (NTA) or normal mouse serum (NMS).
t P < 0.01, Student’s t test.
STEINBERG ET AL
Table 9. Suppression of PWM-Induced IgM Synthesis in Vitro by
Spleen Cells from Mice Treated in Vioo with NTA or N M S or NTA
Absorbed with Thymocytes (Abs-NTA) and Cultured with or without
ZNgcglml Concanavalin A for 36 Hours*
In Vivo
Treatment
Strain
In Vitro
Treatment
Suppression of
IgM Synthesis
NMS
NMS
NMS
NMS
NTA
Abs-NTA
BALB/c
NZB/W
BALB/c
NZB/W
NZB/W
NZB/W
0
0
Con A
Con A
Con A
Con A
-t
-
+
+
+
*One million precultured cells were added to I million BALB/c
spleen cells for the final culture with PWM.
+ = greater than 75% suppression; - = less than 25% suppression.
is hemolytic anemia. Studies performed primarily by
Gershwin demonstrated that repetitive administration
of young NZB thymocytes to NZB mice as they aged led
to a marked reduction in the severity in the autoimmune
hemolytic anemia (13). These studies have been confirmed and extended in our laboratory (14). The suppressive cell is a corticosteroid-sensitive,x-irradiationsensitive thymocyte (Table 10). The lack of effect of xirradiated and freeze-thaw treated cells suggests that
protective immunization (e.g., against viral antigens)
was not the mechanism of action. In addition to suppression of the Coombs’ positivity, young thymocyte
treatment led to retardation of kidney disease and reduction in reticulum cell proliferation (14).
Administration of “Suppressor Cells”
Studies of Suppressor Factors
A logical extension of the above work, which
demonstrated a) a loss of suppressor cells early in life in
New Zealand mice, followed by b) the development of
autoimmunity, was to try to restore suppressor cells and
determine whether or not there would be a favorable
effect on the autoimmune process. Accordingly, female
NZB/W mice were given multiple thymic grafts from
young NZB/W mice. This resulted in marginal increase
in survival (28). Apparently, the grafts led to both improvement (presumably by increasing suppression) and
deterioration (presumably because of the nucleic acid
and thymic membrane antigenic load). We believed that
it might be easier to demonstrate the phenomenon in
NZB mice whose major manifestation of autoimmunity
Suppressor cells act either through direct cell
to cell contact or through some intermediary substance.
Considerable information has been accumulated indicating that both antigen-specific and antigen-nonspecific suppressor cells act through mediators that can
be isolated from cells or released into the medium of
cultured cells (reviewed in ref. 29). Ranney and Steinberg investigated the ability of resting cultures of lymphocytes from NZB/W and control strain mice to generate suppressor factors. Supernates from cultures of
spleen cells from NZB/W mice 2 weeks of age contained
suppressive factors that were released into the culture
medium ( 1 5 ) . Such factors were not produced by NZB/
W spleen cells after 6 weeks of age, whereas, they were
Table 10. Treatment Every 2 Weeks of NZB Mice from 4 Weeks
of Age with Thymocyres (112 Thymus Equivalent) From
NZB Mice I2 Weeks of Age
Table I t . Suppression (-) or Stimulation (+) of Tritiated Thymidine
Incorporation by Unstimulared NZBI W Thymocytes Cultured with
Supernates from NZBl W . C57B1/6 or BALBIc Spleen Cells Cultured
Without Mitogens
~~~~~~~~
~
Treatment
None or medium
Bone marrow
Th ymocytes
Thymocytes,
x-irradiated
Thymocytes,
corticosteroids
pretreated
t
~~
Percent of Mice
with Positive
Direct Coombs’ Degree of
Test at
Glomerular
4.5 Months
Disease
82
85
4*
94
1.75
1.71
I.l6*
NDt
92
ND
Source of Supernate (1 :3 Dilution)
Degree of
Reticulum Cell
Proliferation
Significantly reduced compared to all other groups.
ND-not determined.
1.12
2.67
0.40’
ND
Change, 9%
NZB/W-2 weeks
NZB/W-6 weeks
NZB/W-I0 weeks
NZB/W-20 weeks
-64
BALB/c-2 weeks
BALB/c-6 weeks
BALB/c-10 weeks
BALB/c-20 weeks
-79
- 58
- 52
- 18
-5
+5
+22
ND
C57B1/6-2 weeks
C57B1/6-6 weeks
C57B1/6-10 weeks
C57B1/6-20 weeks
-63
-54
-47
-18
S197
AUTOIMMUNITY IN NZ MICE
Table 12. Degree of Suppression (-) or Stimulation (+) of Tritiated
Table 14. Treatment of Female NZBI W Mice Starting at 20 Weeks
Thymidine Incorporation by Unstimulated 9- Week-Old NZB1 W
Thymocytes Cultured with Supernates from 2- Week-Old NZBl W
Spleen Cells Cultured Without Mitogens
of Age with the Low Molecular Weight Fraction (<lO.OOO) of the
Supernate of Cultures of BALBIc Spleen Cells Stimulated with
Concanavalin A *
Change at Different Dilutions
Dilution of Supernate,%
Fraction of Supernate
Whole supernate
< I ,OOO molecular weight fraction
> 1,OOO molecular weight fraction
I:2
-68
-79
+50
1:4
-44
-67
+54
I :8
+I7
-38
+43
Treatment
Number of
Mice
Survival at
1 Year
48
20
8 (17%)
4 (20%)
24
19 (79%)
1 . None
2. Control CONS, low molecular weight
fraction
3. CONS, low molecular weight fraction
~~~~
produced by spleen cells from control strains at that age
(Table 11).
The factors involved in suppression were found
to be of very low molecular weight (<l,OoO daltons)
(Table 12). We therefore undertook several studies of
such factors. The first was to try t o increase the quantity
of suppressor factor production. This was successfully
accomplished by culture of the spleen cells with Concanavalin A (30) achieving a 100-fold increase. Next,
Reinertsen studied the ability of the suppressive material
to alter in vivo antibody responses to SRBC immunization (16). He found that the immunosuppressive factors
reduced the antibody response to SRBC and also reduced both IgM and IgG antibody producing plaque
forming cells. One of the major mechanisms of immunosuppression was in vivo reduction in spleen cell number
(Table 13). The active principle was of low molecular
weight. We tried to use this low molecular weight material as a therapeutic agent in NZB/W mice. Mice aged
19 weeks, already manifesting autoimmunity with antibodies to DNA and immune complex deposits in their
kidneys, were treated with this low molecular weight
fraction or with control fractions. The mice treated with
the active immunosuppressive principle were found to
have less proteinuria and greater survival (Table 14).
Chi-square: (3) vs (2) = 15.31, P
(3) vs (1) = 26.67, P
* Mice were given 1.5 ml
< 0.001
< 0.001
daily intraperitoneally.
Thus, an antigen nonspecific immunosuppressive fraction of low molecular weight is produced by spleen cells
from young NZB/W mice. The ability to produce this
material is lost prematurely relative to other strains of
mice. Treatment of NZB/W mice with such a fraction
leads to prolongation of survival apparently as a result
of retardation of the autoimmune process.
Ribavirin Studies
Our laboratory has been conducting an extensive
series of studies into the therapeutic efficacy of ribavirin,
a broad spectrum antiviral agent, in NZB/W mice. Initial results indicated that the drug was useful therapeutically in the autoimmune disease of NZB/W mice
(3 1,32). Additional studies suggest that this therapeutic
benefit may occur in the absence of easily demonstrable
immunosuppressive effects. In addition, Williams and
Antonovich have led renal histologic studies that indicate that the drug is capable of preventing electrondense immune complex deposits in the kidney. These
Table 13. Suppression of the 4-Day Direct (IgM) Splenic Plaque-Forming Cell (PFC) Response 10
Intravenous Immunization with 2 X 1P Sheep Erythrocytes by a Less Than 10,OOO Mol Wt/Fraction of
CONS* Given in Vivo
Control-Suppressed
Strain
PFC/Spleen
PFC/IO' Cells
Spleen Cell Number
~~~~
BALB/c
BALB/c X DBA/2
C57B1/6
59,000 (<O.OOOS)t
169,000 (<0.01)
83,000 (<O.OOOS)
110,000(<0.005)
21,000 (<0.025)
31,900 (<0.005)
77,000 (<O.Ol)
87,000 (<0.01)
82,000 (<0.01)
* CONS = Supernate of spleen cells from BALB/c mice cultured for two days with Concanavalin A,
2 rg/ml.
t P value of control versus suppressed determined on the log transformed data by Student's t test.
STEINBERG ET AL
results indicate more than one promising therapeutic
approach for the treatment of autoimmunity in NZB/W
mice.
DISCUSSION
Autoimmunity in NZB mice and their hybrids
appears to be a multifactorial disease. Factors influencing the expression of autoimmunity are a ) genetic, b)
hormonal, c) viral, d ) immunologic, e) nutritional, and
f) biochemical. Most of the factors will ultimately be
found to have a biochemical basis and an effect on the
immune system. In a multifactorial disease, like the
autoimmune disease of NZB/W mice, it is formally
impossible to fulfill Koch’s postulates. Nevertheless, it is
theoretically possible to define the contribution of each
factor by varying it and keeping the others constant.
Perhaps it is more practical to alter one or a limited
number of the factors to ameliorate the disease. Any
factor that is necessary, but not sufficient, for disease
could thus be studied.
These hypotheses lead to questions about the
most important factors. Is the virus necessary? How
many genes are essential? The answer to these questions
is, in part, philosophical. Male NZB hybrids may have
less severe disease than females, but they still have evidence of autoimmunity. If they live an almost normal
life span, is their disease not serious? If the answer is yes
(their disease is not serious), the factors that make
NZB/W females different from NZB/W males are important. These derive from sex chromosomes and may
be largely hormonal. On the other hand, male NZB/W
mice get a perfectly respectable autoimmune illness. Despite protective male sex hormones, or a deficiency in
accelerating female sex hormones, NZB/W males develop antinuclear antibodies and immune complex glomerulonephritis with proteinuria and azotemia. Their
disease is more severe than that of NZB/C3H F, males.
At least one factor in the more severe disease of NZB/W
mice is genetic. Whether or not the genetic factor relates
to viral expression or immunological abnormalities, or
both, remains to be determined. Evidence for an Xlinked immune response gene has been presented for the
control of antibody production to the nucleic acid antigen Poly I . Poly C (33). This concept has been extended
to the response to ssDNA without proper regard for
hormonal consideration (34). The role of an X-linked
immune response gene can be evaluated only by controlling for the hormonal effects of having two X
chromosomes. This can be done by a ) reciprocal crosses,
or b) castration of test animals. The latter is superior if
there is also an important nongenetic maternal factor to
be evaluated. Such studies in our hands suggest that, in
most mice, the role of male sex hormones in retarding
antinucleic acid antibody production is much greater
than any X-linked immune response gene. Despite a lack
of evidence for an X-linked gene controlling anti-DNA
production, there is an autosomal dominant gene which
controls this response. An additional codominant gene
controls production of antibodies to T cells (35).
Biochemical abnormalities include those related
to the adenylcyclase-cyclic AMP-prostaglandin system
and those related to mediators of the immune system.
These may not only interrelate with each other, but also,
indirectly, with thymic and sex hormones.
Despite the many factors involved in autoimmunity in NZB and NZB/W mice, most of the factors
appear to operate through the immune system. A logical
question is “What is the primary defect in the immune
system which leads to autoimmunity?” There may not
be a primary abnormality. In this multifactorial disease,
the hormonal and biochemical factors could act at several steps in the immune response, in more than one
lymphoid organ, and on several cell types.
In NZB mice, evidence has been presented to
support a B-cell defect (36-39) as well as a T-cell defect
(13,14,20,2 1,40-42). T-cell defects in NZB/W mice
(19,23,26,45,46) have been described. Recently Akizuki
in our laboratory has lethally irradiated BALB/c,
DBA/2, CDF,, and NZB/W mice and transferred bone
marrow that was treated with anti-Thy 1.2 and C to
syngeneic and allogeneic mice. Proteinuria and antibodies to DNA were increased and survival was decreased in recipients of NZB/W bone marrow cells (47).
These observations suggest that loss of suppressor cells
may not be the whole cause of autoimmunity; B cells or
other bone marrow cells appear capable of expressing
immune abnormalities.
Perhaps the most unifying concept is that NZB/
W mice have the genetic factors to allow the immune
system to act as if it is excessively stimulated. This
adjuvant-like state could be a direct result of immune
imbalance in favor of immunity rather than tolerance.
Nucleic acids are good adjuvants. In New Zealand mice they are also relatively good antigens (7).
Thus, nucleic acids could act both as immune adjuvants
and as antigens. The source of nucleic .acids for these
purposes could be any dying cells. However, a normal
process, the maturation of erythrocytes, would provide
a continuous source of nucleic acids. Nucleated cell precursors lose their nuclear material in the process of
AUTOIMMUNITY IN NZ MICE
becoming mature erythrocytes, The loss of such material
in the bone marrow could provide both the antigenic
stimulus and the adjuvant to stimulate genetic hyperresponders to produce a variety of antinuclear antibodies. The adjuvant might also shift the balance from
tolerance to immunity with regard to other antigens.
IgG-immune complexes might interact with the Fc receptors of regulatory T cells t o interfere with their functions and further augment immunity.
Another possible cause of an adjuvant-like state
is stem cell infettion by a stimulatory virus. This could
lead to all of the processes described above. Transplacental passage of maternal cells could contribute considerably to either of these processes. Both B cell precursors and prothymocytes might be affected. The
excessive immune reactivity would lead to rapid depletion of the ability of the thymus and bone marrow to put
out uncommitted cells. Suppressor cells might be the
first cell type to be limiting. This would be followed by a
loss of helper T and virgin B cells. Meanwhile, already
committed B cells might replicate excessively and in a
poorly controlled manner.
Some attempt to control a viral infection might
be seen in an antibody response to the virus infected
stem cell. This could lead to NTA formation that would
only accelerate the loss of peripheral recirculating T cells
and also accelerate thymic exhaustion, first of suppressor cells, and then of other T cells. In addition, NTA
appears to preferentially remove suppressor as opposed
to helper cells. The infection of bone marrow stem cells
might accelerate the maturation of prothymocytes.
These events plus the excessive demand for new T cells
would lead to a greater number of cells precommitted
and predifferentiated within the thymus. Thus, for example, Ly 1+2+3+cells would already be differentiated
into Ly 1+2-3- and Ly 1-2+3+ cells within the NZB/W
thymus. Thus, we have a good explanation for the ability of suppressor cells to be activated in 17-week-old
NZB/W mice but unable to recruit new Ly 1+2+3+cells
(functional and in adequate numbers in 5-week but not
17-week mice) to carry out the full process of suppression.
Suppressor substances that might be produced in
response to excessive immune stimulation become relatively deficient between 1 month and 3 months of age.
Later, when the autoimmune process becomes florid, a
form of antigenic competition from excessive autoimmune activity could again lead to suppressor substance
release. It is formally possible that there is a progressive
increase in suppressor function as NZB/W mice age.
Early in life the net effect is to suppress suppressor cells
S199
and later in life to suppress everything. However, from a
functional point of view there is a loss of suppressor
function before a loss of other functions. Further, replacement of suppressive factors from early in life retards autoimmunity (48).
Biochemical and hormonal effects may be mediated through the immune system. This modulation
probably, at least in part, takes place at the level of
cAMP response to antigen and other immune signals.
Abnormalities of T-cell function have been found to
correlate with abnormalities of cAMP responses to antigen (49) as influenced by T cells (50). With regard to
effect on disease, immune complex deposition has been
associated with a switch from IgM to IgG anti-DNA
antibodies which occurs earlier in female than in male
NZB/NZW mice (51,52). NZB parents appear to lack
the gene for the expression of this phenomenon. Nevertheless, it is likely that immune complexes bind to Fc
receptors on suppressor T cells, thereby inactivating
them. This is a mechanism whereby suppressor function
is lost as a result of immune complex disease, thereby
perpetuating the initial process.
Thus, therapeutic intervention could take place
at the level of the biochemical abnormalities, the hormonal abnormalities, the immunologic abnormalities
(cells o r their products), or a viral infection. Hormonal
effects would likely operate through one of the others.
Biochemical treatment is seen in the use of modulators
of the prostaglandin-CAMP system (53). Immunologic
intervention can be antigen-specific as in tolerance to
nucleic acids (54-57) or antigen nonspecific as in restoration of suppressor cells or substances (13,16). Viral
treatment may be a mechanism of ribavirin efficacy. We
should recognize that what works as prophylaxis may
help us to understand pathogenesis. However, we
should not confuse such studies with therapy given after
disease is already present. Finally, nonspecific immunosuppression may reduce the excessive immune process
and allow for a marked improvement in life span (5861).
Immune complex disease of mice caused by a
known virus, LCM, has been successfully treated with
cyclophosphamide (62). Elimination of the host antibody response without change in viral titer led to reduced immune complex deposition. Similar observations have been made in Aleutian mink disease (24,63).
Thus, a favorable response to an immunosuppressive
drug does not imply that a virus or microorganism is
uninvolved. Whether any of the more “specific” therapies will prove t o be better than immunosuppressive
drugs in New Zealand mouse therapy (as opposed to
s200
STEINBERG ET AL
leading to greater understanding of pathogenesis) rem a i n s to b e determined. Ironically, t h e distinction between “specific” (such as suppressor substances a n d antiviral agents) a n d “nonspecific” (immunosuppressive
drugs) therapy m a y soon disappear.
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