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Transfer of spleen cells from young to aging NZB Ф NZW F1 hybrid mice. Effect on mortality antinuclear antibody and renal disease

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TRANSFER OF SPLEEN CELLS FROM
YOUNG TO AGING NZB X NZW Fl
HYBRID MICE
EFFECT ON MORTALITY, ANTINUCLEAR ANTIBODY,
AND RENAL DISEASE
ROBERT E. WOLF and MORRIS Z l F F
Multiple injections of spleen cells from young NZB
NZW F1 hybrid mice with a mean age of 9 weeks were
administered to syngeneic recipients at 10-day intervals
beginning at 3 months of age. The recipient mice had a
delayed appearance of antinuclear antibody, decreased
cumulative incidence of antinuclear antibody positivity up
to 7.3 months of age, lower cumulative mortality up to 8
months of age, and a lesser degree of glomerular sclerosis
at 8 months of age. By 9 months of age no significant
differences in these parameters remained. Transferred
cells were most effective during the 4- to 6-week period
immediately following initiation of injections. The beneficial effects appeared to reflect an early but temporary
suppression of the autoimmune process by the transferred
lymphoid cells.
X
From the Rheumatic Diseases Unit, Department of Internal
Medicine, University of Texas Southwestern Medical School, Dallas,
Texas.
Supported in part by an Arthritis Foundation Postdoctoral
Fellowship, USPHS Program Project Grant No. AM09989 and
USPHS Training Grant No. AM05154.
Robert E. Wolf, M.D., Ph.D.: Arthritis Foundation Postdoctoral Fellow, University of Texas Southwestern Medical School
(presently Assistant Professor of Medicine, University of Texas Southwestern Medical School, and Research Associate, Dallas VA Hospital); Morris Ziff, Ph.D., M.D.: Professor of Medicine, Chief, Rheumatic Diseases Unit, University of Texas Southwestern Medical
School, and recipient, Research Career Award, National Institutes of
Health.
Address reprint requests to Robert E. Wolf, M.D., Rheumatic Diseases Unit, Department of Internal Medicine, University of
Texas Southwestern Medical School, 5323 Harry Hines Boulevard,
Dallas, Texas 75235.
Submitted for publication December 18, 1975; accepted May
20. 1976.
Arthritis and Rheumatism, Vol. 19, No. 6 (November-December 1976)
NZB X NZW F1 hybrid (BW) mice spontaneously develop an SLE-like syndrome ( 1 ) characterized
by the presence of multiple autoantibodies including
anti-DNA and anti-RNA, immune complex glomerulonephritis, diffuse lymphoid infiltrates, and subepidermal
deposits of immunoglobulin (2). Lymphocyte responses
(3-5) and serum thymic hormone activity (6) are depressed, whereas humoral responses are augmented
(3,7-9).
An age-dependent loss of thymus-dependent (T)
suppressor lymphocytes may play an important role in
the pathogenesis of this syndrome. Loss of thymic regulatory control has been suggested by the acceleration of
autoimmune disease (10-12), anti-DNA antibody formation ( I l ) , antibody responses to poly I-poly C (13),
mortality and renal disease in BW mice (IZ), and
Coombs’ positivity in NZB mice (14) after thymectomy
or injection with antilymphocyte globulin or antithymocyte serum. Furthermore, enhancement of antibody
formation to immunization with type I l l pneumococcal
polysaccharide (SSS 111) antigen (15) and graft-versushost ( G V H ) responses (16) have been observed in aging
NZB and BW mice respectively.
The severity of the autoimmune disease in thymectomized (10-12,14) or antilymphocyte globulin
(ALG)-treated (12) BW and NZB mice has been observed to decrease after injection of spleen or thymus
lymphocytes from young syngeneic mice. The effects of
young lymphoid cells on a postulated suppressor cell
deficiency in old BW and NZB mice have been evaluated
(13,16). Implantation of thymus grafts has reversed the
WOLF A N D ZIFF
1354
accelerated autoantibody formation induced in these
mice by thymectomy or antithymocyte serum treatment
(10,l I , 14), and repeated injection of spleen cells or combinations of spleen, thymus, and bone marrow lymphoid cells prevented the early mortality and renal disease observed in BW mice after thymectomy or ALG
injections (12). Both thymocytes and spleen cells from
young BW mice normalized the increased GVH activity
of spleen cells derived from old syngeneic mice ( 1 6 ) .
Few studies have dealt with the effects that transfer of young syngeneic murine lymphocytes has on autoimmune disease not previously accelerated by thymectomy or ALG treatment. Transfer of young spleen
cells or thymocytes prevented the development of antibody to deoxyribonucleoprotein (DNP) in old A/Jax
mice immunized with this antigen (17). Kysela and
Steinberg (18) prolonged the lifespan of BW mice by
multiple young thymus grafts beginning at I month of
age. Recently, Gershwin and Steinberg (19) prevented
development of Coombs' antibody in NZB mice receiving young thymocytes beginning at I month of age.
None of the previous studies evaluated the effects of
prolonged administration of spleen cells on either renal
histopathology or autoimmune disease in female BW
mice. The present studies were undertaken to evaluate
the effects of multiple injections of donor spleen lymphocytes from young BW mice on autoimmune disease,
including renal involvement, in older syngeneic recipient
mice. Spleen cells from young donor mice were repeatedly injected into recipients beginning at 3 months of
age. The effect on time of onset and cumulative incidence of antinuclear antibodies (ANA), cumulative
mortality, and severity of renal disease was studied.
Delayed appearance and decreased cumulative incidence of ANA, decreased early mortality, and amelioration of glomerular sclerosis were observed in mice
treated from 3 to 8 months of age. The beneficial effects
appeared to reflect an early but temporary suppression
of the autoimmune process.
MATERIALS AND METHODS
Mice. Two groups of NZB (female) X NZW (male) FI
(BW) female mice were studied. The recipient group consisted
o f 17 mice which received multiple intraperitoneal injections of
0.7-8.0 X 10' spleen cells (mean: 2.9 X lo') from 6- to I2week-old donors (mean: 9 weeks) in 0.2 ml of medium-I99
(Microbiological Associates, Bethesda, M D) at 10-day intervals from the age of 3 months until sacrifice or death. These
animals were bled by orbital puncture every 3 weeks. Eight
mice were sacrificed at 8 months of age and 5 others at 9
months of age. One mouse died before 8 months of age, and 3
others between 8 and 9 months of age.
Twenty-five control animals were injected with 0.2 ml
of medium-I99 at 10-day intervals from 3 months of age and
were bled every 3 weeks. Eight o f these mice were sacrificed at
8 months of age and 9 at 9 months of age. Six mice died
before 8 months of age, and 2 died between 8 and 9 months of
age.
Lymphoid Cell Suspensions. Spleen cells were collected
by teasing the spleens with forceps in medium-I99 and forcing
the suspensions through a 21-gauge needle three times. Viability was over 90% by trypan blue exclusion.
Antinuclear Antibody. ANA was assayed by using frozen sections of ARS/Sprague-Dawley rat kidney as substrate.
Indirect fluorescent antibody staining was performed with a
fluorescein-conjugated goat anti-mouse IgG antiserum (F: P
ratio: 2.5 pg/mg; protein concentration: 10 mg/mI; Meloy
Laboratories, Springfield, VA) at a 1 : 3 dilution in 0.12 M
phosphate-buffered saline (PBS), pH 7.2. Incubation with
both the mouse sera and the antiserum was performed at room
temperature for 30 minutes and was followed by washing in
PBS. Because undiluted sera from all mice in both groups were
negative at 3 months of age, the development of a reaction
with undiluted serum was considered to be positive. Negative
control sera and saline controls were included, and all slides
were read in a blind fashion.
Renal Histopathology. Kidneys were removed from all
animals at sacrifice. All microscopic sections were read blind.
Renal disease was assessed by light microscopy on formalinfixed kidney sections routinely stained with hematoxylin and
eosin ( H & E ) and Gomori trichrome stains. The degree of
glomerular sclerosis was evaluated on trichrome-stained sections by estimating the percentage of light green staining area
in the glomerulus. The percentage of involvement of individual
glomeruli was tabulated at 5% increments. The mean of readings of 40 glomeruli was recorded for each animal as percentage of glomerulus affected.
Clomerular Deposition of Immunoglobulin. The degree
of renal involvement was also assessed by direct immunofluorescence with monospecific goat antiserum to mouse IgG
(Meloy Laboratories) at a I : 10 dilution. Frozen tissue sections
were incubated with fluorescein-conjugated antiserum for 30
minutes at room temperature and washed with PBS. Scores
from 0 to 3+ were assigned on the basis of amount and
intensity of glomerular staining.
Statistical Evaluation. Overall cumulative incidence of
ANA in control and recipient mice was analyzed by the sign
test (20). The log-likelihood ratio x 2 was used to analyze the
cumulative mortality between 6 and 8 months of age. Nonparametric analyses of scores of glomerular disease using
H & E-stained kidney sections, and of glomerular sclerosis using Gomori trichrome-stained sections, were performed with a
one-sided Mann-Whitney test (21).
RESULTS
Antinuclear Antibody. The onset of ANA positivity (Figure I ) was delayed from 4.5 months of age in the
controls to 5.9 months of age in the recipient mice.
Cumulative ANA positivity was consistently lower in
recipient than in the control mice during the period
between 4.5 and 7.3 months of age, and the sign test
TRANSFER OF SPLEEN CELLS IN NZ MICE
demonstrated that the consistent directional difference
between these two groups of syngeneic mice during this
time period was significant (P = 0.03) (Figure I ) . Once
A N A appeared, progression took place at approximately the same rate in both recipient and control mice,
as judged from the slopes of the curves in Figure 1.
These observations suggest that the main benefit of lymphoid cell transfer was to retard the onset of A N A in the
recipients during a 1-1.5-month period immediately following initiation of spleen cell injections. The lower
A N A titers up to 8 months of age appeared primarily to
reflect this early delay in onset of antibody production.
Mortality. Cumulative mortality data for recipient and control mice are presented in Figure 2. Mortality between 6 and 8 months of age was significantly
reduced in the recipients relative to the control mice (P
< 0.02 by log likelihood x2 analysis of the curves).
Cumulative mortality at 9 months of age, however, was
40% in the control mice and 41% in the recipient mice,
when calculated on the assumption that the mortality
rate among sacrificed animals would have been the same
as that in mice observed between 8 and 9 months of age.
The slopes of the mortality curves indicated a delay in
onset of significant death rates from 6.6 months of age in
control mice to 8 months of age in the spleen cell recipients (Figure 2). These results demonstrated that survival
of BW female mice through 8 months of age was improved by multiple injections of young syngeneic spleen
cells, but that the beneficial effects of lymphoid cell
transfer were vitiated as the autoimmune disease became
more florid. The longer survival of the recipient mice
1355
AGE (months)
Fig 2. Cumulative mortality of 25 B W control mice injected with medium-I99 (0-0) and 17 recipient B W mice (0-0) injected with
spleen cells from young donors (mean age: 9 weeks) at 10-day intervals
beginning at 3 months of age. Because 8 mice from each group were
sacrificed at 8 months. cumulative mortality at 9 months was calculated
on the assumption that the mortality rate among the sacri’jked animals
would have been the same as that in the mice observed beyond 8 months
of age. Comparison of the control and recipient mice between 6 and 8
months of age by the log likelihood x 2 method indicated a Signijcant
difference ( P < 0.02).
also appeared to reflect predominantly the delayed onset
of disease, as noted in the case of the A N A .
Renal Histopathology. Renal histopathologic
changes were studied in both recipient and control mice
sacrificed at 8 and 9 months of age. Diffuse proliferative
glomerulonephritis was observed on H&E-stained sections of kidneys from both groups. The glomeruli were
characterized by varying degrees of endothelial and
mesangial cell proliferation, focal necrosis, hyalinization. and capsular adhesions.
Glomerular sclerosis, determined by the percentage of the glomerulus that stained green with the trichrome stain, was significantly less (Table 1) at 8 months
Table 1. Eflecr of Spleen Cell Transfer on Glomerular Sclerosis in
N Z B X NZ W FI Female Mice.
Age
(months)
AGE (months)
Fig 1. Cumulative incidence of antinuclear antibody in 25 control B W
mice injected with medium-I99 (0-0) and in 17 recipient B W mice
(0-0)
injected with spleen cells from young donors (mean age: 9
weeks) at 10-da-v intervals beginning at 3 months of age. The differences
between 4.5 and 7.3 months were statistically significant by the sign test
IP = 0.03).
8
9
~
Group
No. of
Mice
Percentage of
Glomerulus
Affected
(Mean 5% i SE*)
Control
Recipient
Control
Recipient
8
8
9
5
15.8 f 7.6
2.6 f 0.4
18.2 f 6.6
17.7 f 6.1
~~
* SE: standard error of the mean.
t Mann-Whitney
nonparametric analysis.
3 NS: not significant, P > 0.05.
Pt
<o,oo6
NSS
I356
WOLF AND ZIFF
of age in the recipient group sacrificed at this time (2.6 f
0.4%) than in the control group (15.8 f 7.6%) ( P <
0.006). However at 9 months of age there were no differences between the two groups (Table 1 ). The degree of
glomerular sclerosis appeared to correlate with the severity of disease, because a mean value of 44.0 f 15.9%
was calculated for 3 mice that died before 8 months of
age, and because involvement in the recipient mice increased from 2.6 f 0.4% to 17.7 f 6.1% between 8 and 9
months of age. I t should be pointed out that the differences between treated and untreated mice would have
been larger if the histologic findings in mice dying before
8 months of age had been available for inclusion in the
calculations. The fact that glomerular sclerosis was significantly less in recipient mice than in controls at 8
months but not at 9 months further indicates that the
predominant effect of spleen cell transfer was to delay
the onset of disease and thereby to retard the expression
of the nephritis for 4 to 6 weeks in the recipients.
Glomerular Deposition of Immunoglobulin. The
transfer of spleen cells did not affect the deposition of
IgG in the glomerulus. Mean scores for fluorescent
staining of IgG were 2.2 and 2.0 at 8 months and 2. I and
2.1 at 9 months in the control and recipient mice respectively. The findings at 8 months suggest that deposition
of IgG in the glomeruli preceded the development of
sclerosis.
DISCUSSION
This investigation differs from previous studies of
the effects of young New Zealand mouse lymphocytes
on suppressor activity and autoimmune disease (1012,14,16,17- 19) in two respects: a) Young spleen
lymphocytes were administered over a prolonged period
to aging BW mice that had not had prior thymectomy or
ALG treatment. b) The effect of the transferred lymphocytes on renal histopathology was evaluated. Splenic
lymphocytes were used because suppressor lymphocytes
have been demonstrated in spleen cell preparations
(12,16,17,22-24), and because in previous studies splenic
lymphocytes have effectively suppressed anti-DNP antibodies in A/Jax mice (17), decreased mortality and
renal histopathologic changes in thymectomized and
ALG-treated BW mice ( I 2), and reversed accelerated
graft-versus-host activity in BW mice (16).
The current study demonstrated that injected
spleen cells were most effective in suppressing autoimmune responses during the immediate 4-6-week period
following initiation of treatment, as reflected by the
following observations: The age of onset of ANA positivity was delayed from the age of 4.5 months observed
in the controls to 5.9 months, and there was a similar
delay in the onset of mortality from 6.6 to 8 months of
age. These delays resulted in significant differences between the recipient and control mice in both the cumulative incidence of ANA postivity between 4.5 and 7.3
months of age and mortality between 6 and 8 months of
age.
I n a previous study involving the transfer of
young spleen lymphocytes in BW mice, Kysela and
Steinberg (18) demonstrated only a suggestive increase
in survival of 6-month-old recipients of a single injection
of young spleen cells. The differences in age of the
recipients, 3 months versus 6 months, and the number of
injections, multiple versus single, do not permit direct
comparison of the results in the two studies. A recent
study by Gershwin and Steinberg (19) demonstrated
that multiple injections of young thymocytes from 1
month of age prevented the development of Coombs’
antibody in NZB male mice, whereas spleen cells were
not similarly effective. However a delay in onset of
Coombs’ positivity and lower antibody titers between 5
and 8 months of age were observed by Gershwin and
Steinberg in young spleen cell recipients, although these
differences were not statistically significant when examined by the t test.
In the present investigation, glomerular sclerosis
measured by green staining with Gomori trichrome
stain was also significantly less in the recipient mice than
in the control animals at 8 months of age. The inability
to detect differences in deposition of IgG in the kidney
was not surprising because d l mice had developed ANA
positivity before the time of sacrifice at 8 or 9 months,
and because immunoglobulin was presumably deposited
before the onset of sclerosis. The renal findings were no
doubt influenced by the unavailability of histologic data
from mice dying before sacrifice at 8 or 9 months. In
these mice histologic changes would have been more
severe, had they survived, as is suggested by the greater
degree of glomerular sclerosis found in 3 mice that died
before 8 months of age from which adequate renal tissue
was available for study. Subsequently the disease process appeared to overwhelm the suppression effected by
the injected lymphoid cells and to progress at similar
rates in both recipient and control groups. Cumulative
ANA positivity and glomerular deposition of immunoglobulin were equal at 8 months in both groups, and
cumulative mortality and glomerular sclerosis were
equal at 9 months.
These data lend further support to the concept
that a deficiency of thymic suppressor regulation is an
important factor in the pathogenesis of autoimmunity
and the lupus-like syndrome of BW mice. They demon-
TRANSFER OF SPLEEN CELLS IN N Z MICE
strate for the first time t h e beneficial effects o f multiple
injections of BW spleen lymphocytes from y o u n g BW
d o n o r s on A N A f o r m a t i o n , early mortality, and glomerular sclerosis in t h e kidneys o f aging syngeneic female recipients. T h e results also suggest t h a t t h e immunologic milieu in BW mice between 3 and 4.5 m o n t h s
of age w a s m o r e supportive o f t h e suppressive effects o f
the injected lymphocytes from y o u n g mice t h a n it. was a t
later time periods. T h e y suggest t h a t t h e suppressor T
cells in t h e injected lymphocytes remained active d u r i n g
this period a n d delayed for 4 t o 6 weeks t h e rise in A N A
titer a n d mortality i n t h e recipient mice. It is possible
that in t h e older recipients thymic function w a s inade q u a t e t o s u p p o r t t h e injected T cells, with the result
that A N A titer a n d mortality subsequently rose in parallel with t h e c o n t r o l g r o u p .
T h e presence of lymphocytes t h a t suppress a u toimmunity h a s n o t been previously demonstrated in
peripheral lymphoid tissues such as t h e spleen. D e m o n stration o f this type of suppressor lymphocyte in t h e
present study suggests t h a t peripheral lymphocytes m a y
play a role in preventing the development of a u t o i m munity.
ACKNOWLEDGMENTS
The authors thank Ms. Olivia White for her very capable technical assistance and Ms. Terrie Harris for manuscript transcription.
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