CLINICAL
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
15,
293-300 (1980)
Reduction
of Circulating
Immune Complexes
by
Calorie Restriction
in (NZB x NZW) F, Mice’
S. SAFAI-KUTTI,
Sloan -Kettering
G. FERNANDES, Y. WANG, B. SAFAI, R. A. GOOD,
AND N. K. DAYS
Institute
for
Cancer
Research,
New
York,
New
York
10021
Received August 20, 1979
Levels of circulating immune complexes (CIC) by the Raji cell radioimmunoassay
were analyzed in sera of (NZB x NZW)F, (B/W) mice at four ages (3, 9, 12, and 18
months) on different levels of total food intake (Purina lab chow and two defined diets at
either 20 caYday or 10 Cal/day). In all three groups of animals the CIC levels at 3 months
of age were essentially normal irrespective of dietary intake and increased dramatically
in sera of mice of both sexes at 6 and 9 months. The highest CIC values were recorded
just prior to death at 12 months of age in sera of mice fed Purina lab chow. In contrast,
significantly lower CIC values were observed at 12 months of age on the IO-Cal intake (P
i 0.001). C3 and Ig deposits in the kidney lesions of the mice were strikingly less in those
mice fed the IO-Cal diet than those fed either of the other two diets. This value was also
significantly lower than that of the animals fed 20 cal per day of a defined diet. These
findings suggest that diet may influence lifespan of the shortlived autoimmune-prone
B/W mice by inhibiting the formation and deposition of immune complexes in vital
organs.
INTRODUCTION
(NZB x NZW)F, (B/W) mice are known to be susceptible to autoimmune
diseases and to die at an early age of immune complex nephritis (1,2). Fernandez
et al. (3,4) have shown that the lifespan of these animals is dramatically prolonged
by dietary restriction from weaning and that a low calorie intake has a profound
effect on their immune function. It was also demonstrated that the deposition of
y-globulin
in a capillary distribution
in the glomeruli
of these animals was
markedly inhibited by dietary caloric restriction (4). Indeed, even when restriction
of food intake was initiated long after the onset of autoimmunity,
e.g., at 4-5
months, lifespan was increased, severity of glomerulonephritis
reduced, and deposition of Ig and complement in a capillary distribution minimized (5). Andrews
et al. (6) have recently shown that the levels of circulating immune complexes
(UC) in B/W mice and several other autoimmune strains are regularly elevated. It
therefore appears that an association between CIC and glomerular damage exists.
The purpose of the present study was to investigate the levels of CIC in sera of
B/W mice at different ages on different levels of total food intake and to correlate
the changes in UC with degree of renal damage.
’ Aided by grants from USPHS CA-08748, Al-11843, NS-11457, and Ag-00541, ACS-IM-185, The
Molin Foundation, and the Zelda R. Weintraub Foundation.
’ To whom requests for reprints should be addressed: Memorial Sloan-Kettering
Cancer Center,
1275 York Ave., New York, N.Y. 10021.
293
0090-1229/80/030293-08$01.00/O
Copyright
All rights
0 1980 by Academic Press. Inc.
of reproduction
in any form resewed.
294
Experimental
SAFAI-KUTTI
Animals
MATERIALS
and Diets
ET
AL.
AND METHODS
B/W mice of both sexes were maintained from weaning on three different diets
as described earlier (3). In group I the animals were fed Purina lab chow ad
fibitum; group II was given a defined diet and fed approximately
20 cal per day and
group III was fed a diet restricted to 10 cal per day, but provided vitamins and
minerals in an amount equal to that given mice of group II. The restricted diet is
the one that was previously shown to at least double the lifespan of B/W mice (3).
Sera of the mice of each group were studied at 3,9, and 12 months, and animals on
the low caloric diet also at 18 months of age. Blood was drawn by orbital sinus
puncture, allowed to clot on ice, centrifuged in a refrigerated centrifuge, aliquoted
immediately,
and stored at -70°C until used. Aliquots were thawed only once for
each test.
Raji Cell Radioimmunoassay
To measure serum levels of circulating immune complexes in B/W mice, the
Raji cell radioimmunoassay
previously described by Theofilopoulos
et al. (7) was
modified as follows: Raji cells derived from Burkitt’s lymphoma were cultured in
Eagle’s minimum essential medium (MEM). The cells used in the experiments
were obtained 48 hr after the initiation of the culture. Before each test the viability
of the cells (80-90%) was determined by the trypan blue dye exclusion test.
Preparation
of Aggregated
Mouse
IgG
Chromatographically
purified mouse IgG (Cappel Laboratories,
Cochranville,
Pa. 19330) was dissolved in phosphate-buffered
saline (PBS, pH 7.3) and then
centrifuged at 40,OOOg for 20 min to obtain an aggregate-free solution. The supernatant was removed and incubated in a waterbath at 63°C for 30 min. The solution
containing the aggregates was then centrifuged a second time as above and the
upper one-third of the supernatant was separated frozen in small aliquots at -70°C
in a Revco freezer until used. Just before each analysis the solution was thawed at
room temperature.
Preparation
of lz51 Rabbit
anti-mouse
IgG
The IgG fraction of a rabbit anti-mouse IgG containing antibody (both heavy
and light chains) (Cappel Laboratories) was labeled with lz51 (New England Nuclear, Boston, Mass.) by the chloramine T method as described by McConahey
and Dixon (8).
The specific activity of the lzsI rabbit anti-mouse IgG (4.5 mg/ml) was 0.2
&i/mg
protein. The standard curve for the Raji cell radioimmunoassay
was obtained as follows: 10 ~1 of different dilutions of aggregated mouse y-globulin
(AMG) was preincubated with 15 ~1 of normal human serum (diluted 1:4, 1:6, or
1: 10) as a source of complement.
It was found that the ideal standard curve was
obtained when normal human serum was diluted with normal saline at 1:6. Incubation was carried out at 37°C for 30 min. Next 4.0 x lo6 Raji cells in 50 ~1 MEM
were added and the mixtures incubated at 37°C for 45 min. The cells were then
washed three times with 1 ml of MEM (at 900g for 5 min) in a refrigerated centrifuge. After the final wash, 100 ~1 of lz51 rabbit anti-mouse IgG was added to each
CALORIES
AND
IMMUNE
COMPLEXES
295
tube. The cells were then washed three times again as above and the radioactivity
of the pellet was determined in an autogamma scintillation spectrometer (Packard
autogamma scintillation
spectrometer). All tests were run in duplicate. For the
assay of CIC in mouse sera, the sera from B/W mice were diluted 1:6 with normal
saline and 25 ~1 of this mixture was incubated with 50 ~1 of Raji cells and the test
carried out as described above. Standard statistical methods were used for the
calculation of mean values, standard deviation (SD), and standard error of the
mean (SE). Unless otherwise stated, mean values 2 SE are reported.
Immunofluorescence
Studies
Kidney sections embedded in optimum cutting temperature (OCT) compound
Tissue-Tek II, (Lab-Tek products, Division of Miles Laboratory, Naples, Ill.)
were frozen in a cryostat and stored at -70°C. The frozen kidney sections were
cut into 6 pm sections in a cryostat (- 15 to -2O”C), placed on glass slides, and
stained with fluorescein-conjugated
antisera (antimouse IgG, IgM, IgA, or C3)
(Cappel Laboratories) according to standard procedures (9). These antisera were
previously tested for their specificity by immunodiffusion
and immunoelectrophoresis and diluted 1: 10 to 1:20 in PBS.
Grading of Immunojluorescence
Staining
An arbitrary grading system was adopted with scores of 0 (none), 1 (lowest), 2,
3, and 4 (highest), depending on the observed intensity of the immunofluorescence
staining. In addition to the intensity of the immunofluorescence,
the number of
glomeruli involved were also counted. Each series of slides was examined at least
at two different times. Most often, scores of the two examinations proved to be
identical, but in the event of discrepancy, the grades obtained were averaged.
Statistical Methods
The logarithmic transformation
was used on the CIC values to stabilize the
variances. Analysis of variance methods were used to compare across sex, age,
and diets. The Neuman-Keuls
tests for multiple comparisons were carried out at
each age level to compare across the three diets (10).
RESULTS
Serum Levels of CIC in B/W Mice on Different
Diets
The standard curve obtained in our laboratory for the Raji cell radioimmunoassay for mouse serum is presented in Fig. 1. As shown, this curve resembles
closely the standard curve for Raji cell radioimmunoassay
obtained in our laboratory with human serum. Prior to each experiment, a standard curve was set up.
Figure 2 shows the changes of CIC levels with age of male mice fed three different
diets. At the age of 3 months no difference was observed between the levels of
CIC in mice of the three dietary groups. The mean 2 standard error CIC concentration for mice on standard laboratory chow was 21 2 2 &ml.
The corresponding means for animals on 20 and 10 cal of the defined diet were 37 2 6 and 20 2 5
pg/ml, respectively. All three groups of animals studied at 9 months of age had
elevated values for CIC, and no statistical difference between the mean values
was observed. Thereafter, however, the mice on the diet containing only 10 cal per
2%
SAFAI-KUTTI
ET AL.
9x
cm
6x
3x
4
AGGREGATED
40
200 400
1200
MOUSE
IgG, Hg/ml
FIG. 1. Standard curve established for Raji cell radioimmunoassay.
day showed progressively decreasing values for serum CIC up to the age of 18
months. The highest mean CIC (1217 + 420 pg/ml) was recorded in animals on lab
chow diet at the age of 12 months and at this point the mean CIC in the group of
mice on 10 cal (90 + 17 &ml)
was significantly lower (P < 0.001) than that of
mice fed lab chow. The mean value was also significantly lower (P < 0.01) than
that for animals on 20 cal of the defined diet (518 -+ 179 &ml).
At the age of 18
months this difference between 10 cal and the few survivors on the other two diets
20 Calorie
IO Calorie
--NORMAL
IO
I
2
4
I
6
I
8
IO
12
14
I6
16
I
18
I
20
Age (months)
FIG. 2. Changes of levels of circulating immune complexes with age of male mice fed three different
diets.
CALORIES
AND
IMMUNE
297
COMPLEXES
became still more apparent (P < 0.001). No statistical differences were observed
between CIC levels of mice at any age on lab chow and the 20-cal defined diet. The
median and range of the CIC values and the mean and standard deviation of the
logarithm of the CIC values for each diet, age, and sex are given in Table 1.
Neumal-Keuls
multiple
comparison
tests were based on the mean of the
logarithm of the CIC values.
Figure 3 compares CIC levels obtained in sera of female mice on the three
different diets. At the age of 3 months, all mice on the three different diets had
comparable serum levels of CIC (16 kg/ml). Highly elevated levels for CIC were
recorded in sera of animals on all three diets at the age of 9 months as had been
TABLE
LEVELS
Age
(months)
Male
3-4
9
12
OF CIRCULATING
IMMUNE
1
COMPLEXES
ON DIFFERENT
IN SERA
DIETS
OF MALE
AND
CIC
Diet
(‘.M
n
Standard
20
10
10
10
10
Standard
20
10
Standard
20
10
B/W
FEMALE
MICE
Log CIC
Range
Mean
SD
33
24
16-35
16-80
16-64
3.00
3.48
3.30
0.30
0.50
0.45
10
10
10
392
2%
208
144-6400
176-2240
108-720
6.31
5.97
5.48
1.21
0.82
0.64
10
10
10
840
256
84
304-4800
96- 1760
24- 196
6.75
5.74
4.30fl.b
0.83
1.04
0.70
16-112
3.74
0.79
56- 1440
RX-960
16-56
6&td
5.89d
3.35”,’
1.12
0.62
0.51
2.77
2.77
2.77
-
Median
17
6
40
Standard
20
10
7
7
480
400
21
Standard
20
10
10
10
10
16
16
16
9
Standard
20
10
10
10
10
680
432
280
196- 1920
224-2720
144-960
6.44
6.31
5.71
0.78
0.87
0.60
12
Standard
20
10
10
12
10
632
344
102
196-3200
40-4000
32- 176
6.53
5.79
4.46”,b
0.98
1.40
0.61
15
10
J
16
16- 160
3.65
1.11
15
15-18
15-18
19
Female
3-4
10
9
n Significantly lower than standard diet,
b Significantly lower than 2C-cal diet, P
CSignificantly lower than 20-cal diet, P
d No. of animals used represents a pool
16
16
16
< 0.001, Neuman-Keuls test.
0.01.
0.001.
from three different experimental groups.
P
<
i
298
SAFAI-KUTTI
101
2
ET AL.
4
IO
&
FIG. 3. Changes of levels of circulating
different diets.
4
14 I5
12
kil:“+hs~
immune complexes with age of female mice fed three
observed for the male mice but no statistical difference between the groups was
noted. At the age of 12 months, the mean value for CIC of mice on 10 cal per day
(100 + 16 pg/ml) had declined considerably and was significantly lower (P <
0.001) than the mean for animals on standard laboratory chow (1044 ? 323 pg/ml),
and that of the mice on 20 cal (767 2 331 &ml)
(see Table 1). All female mice on
lab chow and 20 cal died shortly after 12 months and CIC levels of the surviving
female mice on the IO-Cal diet dropped further to 64 2 23 &ml at I5 months of
age.
Glomerular Deposition of Immunoglobulins
and Complement in BIW Mice
These results are summarized in Table 2. Kidney sections of mice on high
caloric intake (17 of 22) showed extensive granular deposits of IgG and C3 (3 -4t)
in the glomerular tufts. In contrast, the deposition of IgG and C3 of mice on the
restricted calorie diet (lo- 15 animals) was minimal and confined primarily to the
mesangium. Kidney sections of 17/26 mice given 20 cal per day demonstrated
2-3t deposition of IgG and C3 in a capillary distribution.
DISCUSSION
In the present report we confirm the findings of Andrews et al. (6) and present
an adaptation of the Raji cell radioimmunoassay
technique of Theofilopoulos
et al.
(7) for analyzing CIC in sera of mice. Our findings reveal as did theirs that in sera
TABLE
HISTOLOGICAL
STUDIES
2
BY IMMUNOFLUORESCENCE
WITH
THREE
OF KIDNEY
DIFFERENT
TISSUE
FROM
MICE
DIETS
IgG and C3
Diet
Total no.
of animals
3-4t
Lab chow
IO cal
20 cal
22
24
26
17
None
None
2-3t
5
6
20
O-It
None
18
6
CALORIES
AND
IMMUNE
COMPLEXES
299
of B/W mice fed laboratory chow ad libitum, the CIC become elevated by 6
months of age and rise progressively thereafter. This increase in CIC is associated
with the deposition of IgG and C3 in a capillary distribution in the glomeruli. We
extended our studies to investigate the influence of calorie restriction on circulating immune complex levels. In the present study serum CIC levels and their
association with glomerular deposition of IgG and C3 were analyzed whenever
possible at four different ages (3, 9, 12, and 18 months) of B/W mice fed on three
different diets (lab chow, defined diet at either 20 or 10 Cal/day). In all three groups
of animals, the CIC levels at 3 months of age were essentially normal irrespective
of dietary intake. Thereafter an increase in serum CIC levels occurred in all
animals. At the age of 9 months, the serum CIC levels were dramatically increased
in mice of both sexes of each dietary group. The highest serum CIC values were
recorded in the animals fed standard lab chow at 12 months of age, i.e., just prior
to death. In contrast, significantly lower CIC values were observed in animals at
12 months of age on the lo-cal intake. Male mice at 18 months of age fed 10 cal
per day showed a further decrease in CIC values while the few surviving mice fed
lab chow or a defined diet with higher calorie intake showed persistent high levels
of CIC. Female mice did not survive to this age. No differences of CIC in mice fed
lab chow or 20 cal per day was evident at any age.
It is of some interest that C3 and Ig deposits in the kidney lesions of the mice on
lab chow ad libitum were apparently greater than those of the group fed the
defined diet at a level of 20 cal per day. The basis for this difference is not
apparent since the CIC levels were not significantly different in the two groups.
The mice fed calorie-restricted
diet had strikingly less deposition of immunoglobulin and C3 in the capillaries of the glomeruli than mice of either of the other two
groups. Nonetheless, deposition of small amounts of Ig could be demonstrated but
it was confined to the mesangium. These findings appear to link the influence of
calorie restriction on length of life and development of severe kidney disease in
B/W mice to an influence on CIC. In prior work we (3, 4) and others (11) have
found that anti-DNA antibody production in B/W mice is diminished by calorie
restriction. By contrast, the amount of virus in the tissues is not diminished by
calorie restriction (1 I). Further, our previous studies showed that dietary manipulations inhibit thymic involution and the decline of certain immunologic
functions with age in B/W mice (4). It is clear from the work of Andrews et al. (6) that
at least some of this decline is due to autoimmunity
directed toward lymphoid
cells. The present observations were especially challenging in that calorie restriction influences the levels of CIC most strikingly or entirely after the animals have
reached 9 months of age. Just why this should be is not immediately apparent and
deserves further analysis. Even though the levels of CIC were similarly elevated
at 6 and 9 months in all dietary groups, the fall of CIC levels in the calorierestricted animals was associated with striking inhibition of deposition of apparent
immune complexes in the glomeruli. Our prior studies (3, 5) established that
glomerular lesions are minimal in the diet-restricted animals at 12-14 months.
These histopathologic and immunologic analyses taken together with observations
recorded herein make it seem likely that either calorie restriction influences the
nature of immune complexes or that renal lesions and immune complex deposits
SAFAI-KUTTI
300
once developed in these mice are reversible.
to clarify this issue.
ET AL.
Further investigation
will be needed
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