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Rapamycin Prolongs Survival and Arrests Pathophysiologic Changes in Murine Systemic Lupus Erythematosus.

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ARTHRITIS & RHEUMATISM Volume 37
Number 2, February 1994, pp 289-297
0 1994, American College of Rheumatology
289
RAPAMYCIN PROLONGS SURVIVAL AND
ARRESTS PATHOPHYSIOLOGIC CHANGES IN
MURINE SYSTEMIC LUPUS ERYTHEMATOSUS
LINDA M. WARNER, L. M. ADAMS, and S. N. SEHGAL
Objective. To evaluate the effects of oral rapamycin (RAPA), a macrolide immunosuppressant that has
been shown to interfere with T cell activation events, on
the course of spontaneous disease progression in the
MRL/MpJ/lpr/lpr (MRLII) mouse model of lupus.
Methods. RAPA treatment (6, 12, or 25 mgkg 3
times per week) was evaluated by monitoring survival
rates, autoantibody levels, and urinary albumin levels.
Additionally, concanavalinA responsiveness, interleukin2 (IL-2) production, lymphoid organ size, and histopathology were evaluated ex vivo.
Results. RAPA prevented the typical rise in antidouble-stranded DNA antibody and urinary albumin
levels and prolonged survival. Spleen and lymph node
sizes were significantly decreased, inflammatory
changes in the lung, liver, kidney, spleen, lymph node,
and thymus were significantly reduced, and T cell
mitogen-stimulated splenocyte proliferation and IL-2
production were restored.
Conchsion. Data from 3 independent experiments demonstrated that RAPA significantly reduced or
prevented many pathologic features of lupus normally
seen in the MRLh mouse, and suggest that RAPA may
be useful as a therapeutic agent in SLE in humans.
The MRL/MpJ/lpr/lpr (MRL/I) mouse is a genetic model of systemic lupus erythematosus (SLE), a
From the InflammatiodBone Metabolism Division, WyethAyerst Research, Princeton, New Jersey, and Sphinx Pharmaceuticals, Durham, North Carolina.
Linda M. Warner, MS: Wyeth-Ayerst Research; L. M.
Adams, PhD: Sphinx Pharmaceuticals; S. N. Sehgal, PhD: WyethAyerst Research.
Address reprint requests to Linda M. Warner, MS, WyethAyerst Research, CN 8000, Princeton, NJ 08543-8000.
Submitted for publication March 23, 1993; accepted in
revised form June 10, 1993.
prototypical autoimmune disease in humans. The autosomal recessive allele, lpr (lymphoproliferation), is
associated with severe lymphadenopathy, early autoantibodies, circulating immune complexes, glomerulonephritis, splenomegaly, arthritic changes (I), pulmonary lesions (2), progressive histopathologic changes
including lymphocytic and monocytic cell infiltrations,
and inflammation and destruction of normal tissue
architecture, all of which contribute to early death (-6
months of age). The wild type, MRL/+/+ (MRL/+),
does not have the recessive lpr gene, and therefore has
a normal lifespan (2 years), with only mild and late
symptoms of arthritis and glomerulonephritis. The
MRL/l mouse is characterized by lymphadenopathy
and proliferation of double-negative lymphocytes
(Thy-1+, a@CD3+, L3T4-, Lyt-2-) (3) that have
lost the normal T cell functions of responsiveness to
concanavalin A (Con A) and production of interleukin2 (IL-2) (4). Therefore, with progression of disease,
there is a growing suppression of mitogenic responsiveness and IL-2 production. This model also contains a B cell component, which is characterized by
polyclonal B cell activation and autoantibody production.
The effects of immunosuppressive agents on
this disease process have been documented ( 5 ) . Although the immunosuppressant cyclosporin A (CSA)
inhibits anti-double-stranded DNA (anti-dsDNA) antibody production in vitro (6), its in vivo effects in the
MRL/l mouse are less consistent. At 100 mg/kg orally,
CSA inhibited proteinuria, but did not extend the life
of the animals compared with that of untreated controls (5). In another study, CSA at a dose of 40 mg/kg
administered intraperitoneally significantly decreased
blood urea nitrogen (BUN) levels and prolonged sur-
WARNER ET AL
290
viva1 in the MRLA mouse (7). A third such study
showed that at 25 mg/kg (orally), CSA neither inhibited albuminuria nor prolonged the lifespan of MRLA
mice (8). Treatment with FK-506 (at 2.5 mgkg intraperitoneally), an immunosuppressive macrolide
with anti-graft rejection properties, prolonged lifespan
and reduced proteinuria in the MRLA mouse, although
it had no effect on anti-dsDNA antibody levels (9).
Rapamycin (RAPA), a macrolide isolated from
the fermentation broth of Streptomyces hygroscopicus
(lO,ll), is a novel immunosuppressant that has been
reported to inhibit allograft rejection in several animal
models (12,13). RAPA has been shown to be more
potent than CSA in inhibiting thymocyte and lymphocyte proliferation, and to interfere with T cell activation events that are CSA insensitive (14). Furthermore, RAPA was shown to be inhibitory in the rat
experimental allergic encephalomyelitis autoimmune
model of multiple sclerosis (15) and in the NOD mouse
model of insulin-dependent diabetes mellitus (16).
We designed several experiments to compare
the effects of oral doses of RAPA with those of CSA in
the MRLA mouse. The clinical course was followed by
monitoring life span, anti-dsDNA antibody levels, and
urinary albumin levels. In addition, Con A responsiveness, IL-2 production, lymphoid organ size, and histopathologic changes were evaluated ex vivo. Our
findings are presented here.
MATERIALS AND METHODS
Experimental design. The first experiment was designed to examine the effect of drug on survival. Experiment
1 included 75 mice (15 per group; age 8 weeks) treated as
follows: naive, vehicle, RAPA 12 mgkg, RAPA 6 mgkg, and
CSA 6 mgkg. Experiment 2 was designed to evaluate drug
effects on anti-dsDNA antibody and urinary albumin levels
as well as survival, and consisted of 72 mice (12 per group;
age 10 weeks) treated as follows: naive, vehicle, RAPA 12.5
mgkg, RAPA 25 m a g , CSA 12.5 mg/kg, and CSA 25
mglkg. Experiment 3 was designed to evaluate immunocompetence and histologic changes, and included 72 mice (12 per
group; age 6 weeks) treated as follows: naive, vehicle,
RAPA 12.5 mgkg, RAPA 25 mgkg, CSA 12.5 mgkg, and
CSA 25 mgkg. This experiment was terminated after 2
months.
Mice. Female MRL/MpJ/lpr/lpr (MRLA) mice (Jackson Laboratories, Bar Harbor, ME) were weighed prior to
the start of the experiment and weekly thereafter. Both urine
and blood were collected prior to the start of the experiment
and at monthly intervals. The mice were bled by retroorbital
sinus puncture and the blood was allowed to clot for 1 hour
at room temperature and then at 4°C overnight. Serum was
processed and stored at -20°C until used.
Age-matched female MRL/MpJ/ +/+ (MRL/+) mice
(Jackson Laboratories) were housed in our barrier facility,
and euthanized at the same time as the MRLA mice. The wild
MRL/+ mice were used as positive controls for the lymphocyte proliferation and IL-2 production experiments.
Drug. RAPA was dissolved in absolute ethanol and
diluted with Cremophor E L (a derivative of castor oil and
ethylene oxide; Sigma, St. Louis, MO) and sterile water to a
final formulation of 8% Cremophor EL, 2% ethanol, and 90%
water. CSA was commercially obtained as Sandimmune
(Sandoz, East Hanover, NJ) for intravenous injection, which
is formulated in a Cremophor/ethanol mixture. This was
diluted in water to give approximately the same concentration of solvents in all doses of both drugs. Freshly made drug
was delivered, at 10 pVgm, by gavage on Monday, Wednesday, and Friday.
Anti-dsDNA radioimmunoassay (RIA). An antidsDNA RIA kit (Diagnostic Products, Los Angeles, CA)
was used to quantitate serum antibodies that bind to doublestranded DNA. The assay was performed according to the
manufacturer’s instructions. The quantity of anti-dsDNA
antibodies was determined by comparison against a standard
curve.
Urinary albumin. The Multibumin Kit (Exocell, Philadelphia, PA) was used to quantitate albumin found in the
urine. The standard assay procedure was altered slightly to
give a standard curve from 250 pg/ml to 2,500 pdml. Briefly,
each standard was diluted 1:20 in sterile water, and 100 pl of
the standard or test urine was added to each well of a 96-well
plate. Two hundred microliters of bromocresol green was
added to each well. The absorbance was read at 630 nm. The
quantity of urinary albumin in each sample was determined
from the standard curve.
Histology. The heart, lung, trachea, 2 inguinal and 2
axillary lymph nodes, the spleen, liver, both kidneys with
adrenals, and the thymus were removed and immediately
placed in 10% formaldehyde. Samples of these tissue were
processed for histology by American Histolabs, Gaithersburg, MD. Tissue sections were stained with hematoxylin
and eosin, and evaluated in a blind manner by a consulting
pathologist (Raj Sharma, DVM, MS, Wilmington, DE).
Changes were graded 0 4 , where 0 = none, 1 = minimal, 2
= mild, 3 = moderate, and 4 = marked.
Splenocyte prolieration. The following method, as
described in part by Strong et al(17) and Janossey et al(18),
was used for the ex vivo spleen cell proliferation assay.
Spleens were removed and pressed through a stainless steel
500-mesh screen to produce a single-cell suspension. Erythrocytes were lysed by a 4-minute incubation in 0.83%
(weightholume) ammonium chloride, and the cells were
immediately washed twice with RPMI 1640 medium. The
splenocytes were resuspended to a concentration of 5 x lo6
cells/ml in RPMI 1640 medium containing 10% fetal calf
serum, 100 units/ml penicillin, 100 pg/ml streptomycin, 2
mM L-glutamine, 0.1 mM non-essential amino acids, 1 mM
sodium pyruvate, and 50 ph4 2-mercaptoethanol. Cells were
incubated for 72 hours at 37°C in a 5% CO, atmosphere in
%-well microtiter plates at a concentration of 5 x los
cells/well.
Mitogens were diluted to the appropriate concentrations in the incubation medium, and added to the wells at the
beginning of the incubation period to give a final concentration of 2.0 pg/ml Con A, 10 d m l lipopolysaccharide (LPS),
10 pdml phytohemagglutinin (PHA), or 10 ndml phorbol
29 1
RAPAMYCIN PROLONGS SURVIVAL IN MURINE LUPUS
myristate acid (PMA) in a final volume of 0.2 ml. Spontaneous proliferation (without mitogen) was also assessed. Proliferation in wells was assessed by incorporation of tritiated
thymidine (1 pCi/ml, final concentration) during the last 18
hours of incubation. Splenocytes from 6 animals per group
were separately analyzed in culture.
Splenocyte IL-2production. Cultures of spleen cells
from the same animals used for the proliferation studies were
established in the same manner as described above, using
only the mitogen Con A. Cells were incubated for 24 hours at
37°C in an atmosphere of 5% CO, in 96-well microtiter
plates. Supernatants were collected (600 plhample) and
assayed for IL-2 content using IL-2-dependent CTLL-2 cells
as described by Gillis and Smith (19). Briefly, healthy
CTLL-2 cells were harvested, washed twice to remove
residual IL-2, and resuspended in fresh assay medium at 5 x
lo4 cells/ml. The wells of a 96-well microtiter plate were first
filled with 100 pl of sample to be tested (done in triplicate).
The standard curve was set up by filling the appropriate
wells with 100 pl of assay medium, and then 100 pl of
recombinant human IL-2 (rHuIL-2) was added to the first
well of each column (also done in triplicate). Two-fold serial
dilutions were made down the plate, and the last 100 pl was
discarded. The standard curve started at 50 units/ml final
concentration of rHuIL-2, with 8 two-fold dilutions.
When all samples and controls were in place, 100 pl
of cell suspension was added to each well. The cells were
incubated overnight at 37°C in 5% CO, and then pulsed with
tritiated thymidine (final concentration 1 pCi/ml). Following
an additional 8-hour incubation, the cells were harvested
onto glass-fiber filters, which were then deposited into
scintillation vials. The vials were filled with 2 ml of Aquassure scintillation fluid (Dupont, Boston, MA) and counted on
a beta counter (counts per minute were recorded).
Statistical analysis. The Mantel-Haenszel test was
used to evaluate survival rates. The survival rate for each
treatment group was determined by comparing the slope of
the line corresponding to the deaths of the animals over time
with that of the vehicle control. P values were calculated,
and a trend test was performed. A simple linear regression
model, using the untransformed RAPA dose as the independent variable and the lifespan as the response, was used to
analyze the data from both the first and the second experiments (separately and combined) to estimate a median
effective dose (ED,,). The two survival studies were compared with each other for 2 groups: RAPA 12 mg/kg or 12.5
mgkg and vehicle.
For evaluation of the anti-dsDNA antibody and
urinary albumin data, a t-test on the mean log difference per
group was performed for each month. In addition to analyses
of variance (ANOVA), the Student-Neuman-Keuls test was
performed for comparisons of treatment effects on the organ
measurements and proliferation studies. Tests for linear
trend with dose increases were performed for each type of
stimulus (mitogen) for RAPA and CSA, using vehicle as a
zero dose.
Analyses of the histomorphology data were performed only for those conditions for which 5 or more mice
experienced changes. Analyses which assumed a repeated
measures design were performed for the lung, liver, and
kidney data, since 2 or 3 abnormal conditions were reported
for each of those organs. Separate one-way ANOVA were
--
@------
Naive
-0 Vehicle
+-----A
-5
m
$
a
-+
+---.--O
A4A-A.l. -!
-------O
RAPA 25
RAPA125
CsA 25
CsA 12.5
50
40
30
20
10
0
0
50
100
150
200
250
300
350
400
450
500
550
Day
Figure 1. Rates of survival of MRLh mice (experiment 2). Twelve
mice per group (age 10 weeks) were given oral doses of vehicle (8%
Cremophor EL, 2% ethanol), rapamycin (RAPA), or cyclosporin A
(CsA) 3 times per week. On day 517 (no surviving mice), the survival
rates of each group were compared with that of the vehicle control
group (134 days), yielding the following values: naive mice 90 days
(P= 0.23), RAPA 12.5 mg/kg 155 days (P= 0.03), RAPA 25 mgkg
237 days (P= 0.001), CsA 12.5 m a g 126 days (P= 0.96), and CsA
25 mg/kg 106 days (P= 0.14).
performed for spleen, adrenals, lymph nodes, and thymus
data. Pairwise comparisons to the vehicle were performed
using the least significant difference method.
RESULTS
Survival evaluation. In the first experiment,
RAPA at 12 mg/kg significantly prolonged median
survival (283 days) compared with the vehicle control
(162 days) (P = 0.046), while neither RAPA at 6 mg/kg
(237 days; P = 0.115) nor CSA at 6 mg/kg (171 days;
P = 0.574) had a significant effect on survival. However, a significant dose-response relationship was
found in the survival of the mice treated with RAPA
(P = 0.05).
In the second experiment, RAPA at 12.5 mg/kg
and at 25 mg/kg significantly increased survival (P =
0.03 and P = 0.001, respectively) in a dose-dependent
manner compared with vehicle control. CSA at 12.5
mg/kg and at 25 mg/kg was without significant effect
(P = 0.96 and P = 0.14, respectively). The survival
curves are depicted in Figure 1 .
Survival rates in the 2 different experiments
were not significantly different, either for similar
RAPA doses or for vehicle control (P= 0.7 and P =
0.8, respectively). When we combined the like treatment groups of the 2 experiments and compared these
against each other, the difference was significant (P =
0.02), indicating that up to day 128, the survival rates
were similar in both studies for the same groups. The
oral ED,, values for the first two experiments were
WARNER ET AL
292
Table 1. Mean serum anti-dsDNA antibody and mean urinary
albumin levels in MRLA mice, by treatment group*
Anti-dsDNA
(unitdml)
Treatment
Control
Naive
Vehicle
RAPA
12.5 mg/kg
25 mg/kg
CSA
12.5 mg/kg
25 mg/kg
Albumin
(CLg/ml)
Prestudy
At 2
months
Prestudy
Last
sample
34
53
211t
183-t
5%
540
3,406t.
3,253$
28
49
68
63
786
974
819
764
58
28
91t
240t
699
837
712
764
* Groups of 12 10-week-oldmice were treated 3 timedweek for their
lifespans with oral doses of vehicle (8% Cremophor EL, 2% ethanol), rapamycin (RAPA), or cyclosporin A (CSA) (see Materials and
Methods for further details). Serum anti-double-stranded DNA
(anti-dsDNA) antibodies were determined by radioimmunoassay (at
age 18 weeks, 2 months of study), and urinary albumin levels were
determined by bromocresol green-binding assay (ages varied).
t Mean log difference significantly different from zero (see text for
details).
$ P < 0.01 versus prestudy values.
estimated at 16.4 mg/kg (95% confidence limits - 1.9,
34) and 16.0 mg/kg (95% limits 4.3, 28), respectively.
The combined study EDso was estimated at 16.1 mglkg
(95% limits 8.6, 23.6).
Anti-dsDNA antibodies. There were no significant differences in the anti-dsDNA antibody levels
among all the mice prior to the start of the study.
When the interaction between treatment and time was
evaluated within groups, a significant difference was
obtained (P = 0.01). Therefore, the anti-dsDNA antibody levels for each mouse were evaluated on a
monthly basis and compared with the corresponding
values for that mouse at the start of the study. These
monthly P values were averaged for each group,
subtracted from the averaged prestudy values, and
compared to zero, giving the following overall P
values: naive = 0.001, vehicle = 0.005, RAPA 12.5
mg/kg = 0.08, RAPA 25 mglkg = 0.55, CSA 12.5
mg/kg = 0.001, and CSA 25 mg/kg = 0.001. Only those
animals treated with RAPA maintained anti-dsDNA
antibody levels similar to their prestudy levels. All
others were significantly elevated with time. Table 1
shows the mean peak anti-dsDNA antibody levels
obtained at age 4.5 months compared with the mean
prestudy values.
Urinary albumin levels. There were no significant differences in the urinary albumin levels among
all the mice prior to the start of the study. When the
interaction between treatment and time was evaluated
within groups, a significant difference was obtained
(P = 0.02). Therefore, the urinary albumin values were
treated as the anti-dsDNA values (see above). The
following overall P values for each group were obtained: naive = 0.001, vehicle = 0.001, RAPA 12.5
mglkg = 0.40, RAPA 25 mg/kg = 0.57, CSA 12.5
mg/kg = 0.11, and CSA 25 mg/kg = 0.93. Thus,
Table 2. Prevalence of histopathologic findings in MRLA mice, by
treatment group*
~
~
Control
RAPA
CSA
~~
Tissue, feature
Lung
Focal peribronchial MNC
infiltrate
Focal perivascular MNC
infiltrate
Focal hemorrhage
Abscess
Congestion
Focal thrombosis
Focal pneumonitis
Focal edema
Liver
Focal periportal inflam.
cell infiltrate
Focal perivascular inflam.
cell infiltrate
Focal inflammation
Focal vasculitis
Focal bile duct
hypertrophy
Autolysis
Heart
Focal pericarditis
Spleen
Lymphoid hyperplasia
Kidney
Focal vasculitis
Focal pyelitis
Focal glomerulonephritis
Focal interstitial nephritis
Focal cyst, tubule
Adrenal gland
Focal vacuolation, cortex
Lymph node
Lymphoid hyperplasia
Focal necrosis
Thymus
Lymphoid hyperplasia
Autolysis
Naive Vehicle 1 2 3 25 12.5 25
12
7
5
0
4
1
12
8
6
2
10
I1
1
0
0
1
1
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
1
1
0
0
0
0
0
0
1
0
4
8
2
2
8
7
5
4
1
1
2
1
1
2
0
0
0
0
1
0
2
1
1
0
0
0
0
0
0
0
0
0
1
0
0
0
1
0
0
0
1
0
12
12
0
0
12
12
12
12
5
12
10
3
7
0
5
0
12
I1
0
2
1
12
12
2
1
0
4
0
0
1
0
0
9
7
0
4
0
10
11
2
4
11
9
12
0
12
0
0
1
12
12
0
1
0
llt
12
0
0
Ot
0
llt
0
12
0
0
2
8
0
1
* Groups of 12 6-week-old mice were treated 3 timedweek for 2
months with oral doses (in mg/kg) of the indicated compounds (see
Materials and Methods for details). MNC = mononuclear cell;
inflam. = inflammatory. See Table 1 for other definitions.
t Tissue sections from only 11 mice were available for examination.
293
RAPAMYCIN PROLONGS SURVIVAL IN MURINE LUPUS
urinary albumin levels of non-drug-treated animals
increased significantly over time. However, those animals dosed with either drug maintained urinary albumin levels not significantly different from those prior
to the start of the study. Table 1 shows the mean
urinary albumin levels last obtained and those obtained prestudy.
Histologic features. The incidence of histomorphologic findings in the third study are presented in
Table 2. The pairwise comparisons of RAPA (both
doses) and vehicle treatment for the kidney vasculitis,
pyelitis, and nephritis, spleen, lymph node, and thymus lymphoid hyperplasia, and focal vacuolation of
the adrenals gave P values of <0.001. The pairwise
comparison of RAPA (25 mg/kg) versus vehicle for the
A
B
Figure 2. Histomorphologic appearance of the lung in 14-week-old
naive and rapamycin (RAPAFtreated (25 mg/kg) MRLh mice. A,
Tissue from a naive mouse, showing perivascular, peribronchial,
and intrabronchial inflammatory cell infiltration. B, Tissue from a
RAPA-treated mouse appears
histologically
__
- “unremarkable.”
(Original magnification X 40.)
A
B
Figure 3. Histomorphologic appearance of the kidney in 14-weekold naive and rapamycin (RAPAhtreated (25 mg/kg) MRWl mice.
A, Tissue from a naive mouse, showing perivascular inflammatory
cell infiltration. B, Tissue from a RAPA-treated mouse appears
histologically “unremarkable.” (Original magnification x 40.)
peribronchial and perivascular mononuclear cell infiltration in the lung and for periportal inflammatory cell
infiltration in the liver gave P values of 0.02 and 0.03,
respectively.
Focal peribronchial or perivascular mononuclear cell infiltration in the lung is a common finding
in this strain of mice. In the present study, 100%
incidence in the naive MRLA group was observed.
RAPA (25 mg/kg) significantly reduced both focal
peribronchial mononuclear cell infiltration (P = 0.02)
and focal nerivascular mononuclear cell infiltration
(P = 0.02j(~eeFigure 2). CSA (12.5 mg/kg and 25
mg/kg) significantly worsened the focal perivascula(P = Oo0l2 for ep
mononuclear
dosage).
294
WARNER ET AL
Table 3. Effects of RAPA and CSA on MRLA mouse spleen weight and lymph node diameter
Lymph node diameter (mm)
Treatment
Control
Naive
Vehicle
RAPA
12.5 mgikg
25 mg/kg
CSA
12.5 mdkg
25 mdkg
Inguinal
Spleen
weight (gm)
Left
Axillary
Right
Left
6.5 f 0.6
4.9 2 0.5
10.8 f 0.7
9.3 f 0.7
0.41 f 0.07
0.28 f 0.03
6.9
5.0
0.19
0.14
3.0 f 0.3%
2.9 f 0.2$
2.4 f 0.3%
2.7 f 0.2$
7.9 f 0.9t
6.9 0.4t
5.6 f 0.6
5.8 f 0.6
f 0.01%
f 0.00%
0.38 f 0.03
0.30 f 0.02
f 0.6t
f 0.5
*
3.5
3.9
f 0.4$
* 0.2$
10.3 f 0.8
10.0 f 0.4
Right
f 0.7
10.0 2 0.6
11.0
4.1
4.1
-t
f
0.3%
0.2$
10.6 f 0.8
9.9 2 0.6
* Groups of 12 mice were treated 3 timedweek for 2 months with oral doses of the indicated
compounds (see Materials and Methods for details; see Table 1 for definitions of abbreviations).
Values are the mean f SD.
t Significantly increased compared with vehicle control ( P 5 0.05).
% Significantly decreased compared with vehicle control (P5 0.05).
RAPA (12.5 mg/kg and 25 mg/kg) significantly
reduced focal periportal inflammatory cell infiltration
in the liver (P = 0.03 for each dosage). Lymphoid
hyperplasia, characterized by an increase in the number of lymphoid cells and/or size of lymphoid follicles,
was seen in the spleen, lymph nodes, and thymus of
the naive, vehicle-treated, and both CSA-treated
groups. The severity of this change was similar in all
affected groups. RAPA (12.5 mg/kg and 25 mg/kg)
significantly reduced this lymphoid hyperplasia (P =
0.001 for each tissue).
In the kidney, CSA (25 mg/kg) significantly
exacerbated focal vasculitis (P = 0.05), characterized
by inflammatory cell infiltration around blood vessels,
and focal pyelitis (P = 0.03), characterized by inflammatory cell infiltration in the pelvis of the kidney.
RAPA (12.5 and 25 mg/kg) significantly reduced both
focal vasculitis and focal pyelitis (P = 0.001 for each
finding) (see Figure 3). Interstitial nephritis was significantly reduced by both RAPA (P = 0.01 for each
dosage) and CSA (P = 0.01 for each dosage). The
incidence of focal vacuolation in the cortex of the
adrenals, a common finding in this strain of mice, was
significantly reduced by RAPA (P = 0.001 for each
dosage).
Organ size. Significant differences between
treatments (P < 0.001) were noted for spleen weights.
Spleen weights for the RAPA 25 mg/kg group were
significantly lower than spleen weights for the RAPA
12.5 mg/kg group, which, in turn, were significantly
lower than those for the other 4 groups (P5 0.05 for
each comparison) (see Table 3).
The two RAPA-treated groups had significantly
smaller lymph node diameters than the other groups
(P = 0.05). Interestingly, for all treatment groups, the
axillary nodes were larger than the left inguinal nodes,
which were larger than the right inguinal nodes.
RAPA, at both doses, significantly reduced the size of
both the inguinal and axillary nodes (right and left)
compared with the vehicle-treated group (P s 0.05 for
each comparison) (see Table 3).
Splenocyte proliferation. RAPA (12.5 mg/kg and
25 mgkg) significantly restored splenocyte responsiveness to the T cell mitogens Con A and PHA, when
compared with the vehicle response (P < 0.05 for each
dosage). This restored responsiveness approached
normal levels for Con A (38,118 cpm versus 48,818
cpm for Con A-stimulated MRL/+ splenocytes), while
PHA approximated 40% normal activity (22,688 cpm
versus 59,114 cpm for PHA-stimulated MRL/+ splenocytes). In contrast, CSA had no significant effect on
Con A- or PHA-stimulated splenocyte proliferation
(see Table 4).
Only RAPA at 25 mg/kg significantly increased
the splenocyte response to the B cell mitogen LPS,
compared with the vehicle-treated group (P I0.05).
However, the vehicle control group had a significantly
greater LPS response than did the naive group. RAPA,
at both doses, significantly increased PHA-induced
proliferation compared with the vehicle group (P 5
0.05). It appeared that the splenocyte responsiveness
of the naive and vehicle-treated groups to PMA was
not impaired, since they were not significantly different from the MRL/+ response (see Table 4).
Interestingly, spontaneous splenocyte proliferation was significantly increased (P< 0.05) in animals
dosed with either RAPA or CSA at 25 mg/kg and this
response was significantly greater (P C 0.05) than the
RAPAMYCIN PROLONGS SURVIVAL IN MURINE LUPUS
Table 4.
295
Effects of RAPA and CSA on MRLA mouse splenocyte proliferation ex vivo*
Mitogen stimulation
Treatment
No mitogen
Con A
Control
Naive
Vehicle
RAPA
12.5 mg/kg
25 mg/kg
CSA
12.5 mg/kg
25 mg/kg
1,536 f 191
2,536 f 389$
27,251
38,118
1,128 f 56
2,215 f 275$
5,744
7,136
Normal MRL/+ mice
1,269 f 102
748
1,051
f
f
117
145
3,584 f 813
6,038 rt 730
f 2,096$
LPS
PHA
PMA
23,543 2 4,048t
33,186 f 2,104
7,013 2 1,729
10,144 t 1,470
3,629
3,655
2
2
345
290
f
2,754$
41,729 f 1,498
45,884 f 1,839$
24,318 4 1,964$
22,688 4 1,819$
4,492
5,105
f 325$
f 1,293
f
k
1,367
1,065
31,745 f 1,843
39,908 f 1,341
8,780 f 2,244
16,300 2 3,200
3,487
4,329
f 181
f 270
44,925 f 1,998$
59,114 4 3,461$
4,453 2 438
48,818 f 4,240$
* Groups of 12 mice were treated 3 timedweek for 2 months with oral doses of the indicated compounds. Six mice from each group were
randomly selected for evaluation of 72-hour splenocyte proliferation in the presence and absence of mitogen, as determined by uptake of
tritiated thymidine (see Materials and Methods for details). Values are the mean f SEM cpm for 6 animals per group. Con A = concanavalin
A; PHA = phytohemagglutinin; LPS = lipopolysaccharide; PMA = phorbol myristate acetate (see Table 1 for other definitions).
t Significantly decreased compared with vehicle control (P5 0.05).
$ Significantly increased compared with vehicle control (P5 0.05).
MRL/+ response (Table 4). RAPA significantly affected spontaneous as well as mitogen-induced (except
PMA) splenocyte proliferation in a dose-dependent
manner, giving the following P values for linear trend:
P < 0.004 for spontaneous, Con A, LPS, and PHA,
and P = 0.088 for PMA. No dose-response relationship between CSA and either Con A-, LPS-, PHA-, or
PMA-induced splenocyte proliferation was seen (P >
0.05).
IL-2production. The ability to produce IL-2 in
response to Con A stimulation was significantly restored (P 5 0.05 for each dosage) only in those animals
dosed with RAPA. This correlated to those animals with
restored T cell mitogen responsiveness (see Table 5).
DISCUSSION
The prophylactic effect of oral RAPA on SLE in
MRLA mice is clearly evident. RAPA, when delivered
to very young MRLA mice, had a profound effect on
many pathologies normally present in these mice.
Spleen and lymph node sizes were significantly decreased. T cell mitogen-stimulated splenocyte proliferation and IL-2 production were restored. Inflammatory changes in the lung, liver, and kidney, focal
vacuolation in the adrenal cortex, and lymphoid hyperplasia in the spleen, lymph node, and thymus were
significantly reduced. CSA at the same doses did not
significantly reduce any pathologic changes except the
incidence and severity of interstitial nephritis. Unlike
both CSA and FK-506, RAPA prevented the rise in
anti-dsDNA antibody titers seen with disease progression in this model. These results reproduce the work
by Mountz et a1 (7) and Berden et a1 (8), which
demonstrated that CSA did not inhibit anti-dsDNA
antibody production in vivo.
RAPA, as well as CSA, prevented the rise in
urinary albumin levels normally seen in this model and
viewed as an indication of glomerulonephritis. These
CSA results contrast Berden's findings that CSA (25
mgkg orally) did not reduce albuminuria levels when
compared with control MRLA animals (8). However,
both Bartlett et a1 (using 100 mg/kg oral CSA) and
Mountz et a1 (using 40 mg/kg intraperitoneal CSA)
Table 5. Effects of RAPA and CSA on MRLA mouse splenocyte
production of IL-2 ex vivo*
IL-2 production
Treatment
Control
Naive
Vehicle
RAPA
12.5 mgkg
25 m a g
CSA
12.5 mgkg
25 mg/kg
Normal MRL+/+ mice
cpm
Units/ml
2.706
3,531
f
f 610
0.191
0.238
f 0.031
f 0.035
9,166
8,317
f 602t
f 1,516t
0.562
0.535
f 0.037t
rt 0.106'r
2,573
2,438
f 687
0.174
0.168
f 0.042
f 0.032
546
f 485
13,775 f 1,273t
0.955 2 0.144'r
* Groups of 12 mice were treated 3 timedweek for 2 months with
oral doses of the indicated compounds. Splenocytes were incubated for 24 hours with concanavalin A, and the interleukin-2
(IL-2) content in the supernatants was determined by CTLL-2
bioassay (see Materials and Methods for details). Values are the
mean f SEM. See Table 1 for other definitions.
'r P < 0.05 versus vehicle.
WARNER ET AL
reported an inhibition of glomerulonephritis, as evidenced by decreased albuminuria or BUN levels (5,7).
Amelioration of anti-dsDNA antibody and urinary albumin production by RAPA correlated with
prolonged survival in the MRL/1 mouse. The importance of these findings is in the clinical relevance of
these pathologies for humans with SLE. For example,
in humans, T cells are hypoactive and deficient in IL-2
production following PHA stimulation ex vivo (20).
Also, most SLE patients present chronic lymphoid
hyperplasia (21). In addition, anti-dsDNA antibodies
correlate with disease exacerbations in humans (22).
Lastly, renal failure secondary to lupus nephritis is a
common cause of morbidity in humans with SLE (21).
RAPA ameliorated several of the parameters of disease activity in the MRLA mouse (i.e., anti-dsDNA
antibodies, nephritis, etc.), which resemble some of
the strong clinical features of human lupus.
Data from 3 independent experiments demonstrate that RAPA significantly reduced or prevented
many pathologies of lupus in the MRL/l mouse. RAPA
was clearly more efficacious than CSA in this model.
The lime of initiation of RAPA treatment may be
important in shaping the survival curves of the MRLA
mouse. Prophylactic treatment with RAPA (initiation
of drug at 6 weeks of age) appears to result in optimal
survival of the MRLA mouse, when given at the
aforementioned dosing regimen. Therapeutic effects of
RAPA need further study.
Although the mechanism(s) of action of RAPA
in this model is not yet known, its effects in vitro
suggest modulation of T cell-mediated events (23).
Spontaneous lymphoid hyperplasia and/or autoimmunity are not detectable in the MRL/l mouse prior to 6
weeks of age, during which time immunocompetence
is demonstrable (24). However, after this age, an
unusual subset of lymphocytes of a T cell lineage
(L3T4-, Lyt-2-) is involved in progressive lymphadenopathy, immunoincompetence, and autoimmunity
(3). The ability of RAPA to inhibit T cell activation
may function to inhibit the activation and proliferation
of these double-negative T lymphocytes. This could
subsequently inhibit those pathologic changes associated with the presence of these proliferating T cells,
and therefore maintain immunocompetence while prolonging survival of the MRLA mouse. In a collaborative study (Yocum D, University of Arizona, Phoenix:
personal communication), only those animals treated
with RAPA showed a decrease in the double-negative
phenotype of mononuclear cells isolated from the
spleen, lymph node, and thymus.
By preventing the expansion of these double-
negative immature cells, RAPA may restore a normal
ratio between T cell subpopulations; a beginning step
in restoring immunocompetence. By normalizing the
amount of lymphokines produced and their availability
to functional T cells, restoration of both a proliferative
response to T cell antigens and concomitant IL-2 production, which are usually suppressed in these animals
(25), may result. If RAPA functions to inhibit the proliferation of these double-negative T cells, which spontaneously produce B cell dserentiation factor (26), the
stimulus for polyclonal expansion of B cells would be
significantly decreased. Lastly, the L3T4+ T cell subpopulation found in MRL/I mice with renal vasculitis,
which appears to spontaneously proliferate and be
autoreactive to the Ia molecule (27), could be inhibited
by RAPA, thus providing a direct suppressive effect
on autoimmunity in this lupus model.
The effect of RAPA in vitro also suggest modulation of non-T cell areas of immunity involving
macrophages and B cells. For example, RAPA directly
inhibits pokeweed mitogen-induced proliferation and
IgG production (28) by B cells. In addition, nanomolar
concentrations of RAPA inhibit IL-I production by
resident murine macrophages (Adams L: unpublished
observations). Also, RAPA blocks phosphorylation of
p70 S6 kinase in a lymphokine signal-transduction
pathway (29). RAPA had a profound effect on the
polyclonal B cell component of this disease and on
autoantibody production. RAPA may inhibit the signal
transduction of lymphokines which are critical in this
pathology. Alternatively, RAPA may interfere with the
stimulus for activating autoreactive T cells in MRLA
mice, which is derived from la-bearing B cells (30,31). In
addition, since macrophages secrete IL-Ip as they bind
and process antigens (32), RAPA may inhibit this process involving self antigens in autoimmunity.
In summary, RAPA’s effects on non-T cell
areas may include a direct inhibition of B cell proliferation or antibody production, interference of signal
transduction pathways for important B cell growth
factors, prevention of the stimulus provided by
antigen-presenting cells (B cell and/or macrophages) to
the autoreactive T cells, or inhibition of the secretion
of some lymphokines. Effects at any or all of these
non-T cell areas of immunity could decrease the
production of autoantibodies and immune complex
formation. Consequently, the incidence and seventy
of glomerulonephritis, a major contributor to death in
these animals, would be reduced. It is likely that
RAPA affects multiple mechanisms of pathogenesis in
this model. Since these data show that the amelioration of histopathologic changes and normalization of
RAPAMYCIN PROLONGS SURVIVAL IN MURINE LUPUS
biochemical and immunologic parameters are correlated
with prolonged survival in RAPA-treated MRLA mice,
RAPA might be useful as a therapeutic agent for SLE in
humans. Therefore, the conclusion of the ongoing phase
I safety studies with RAPA, which will indicate its
therapeutic index in humans, will be critical when
considering this immunosuppressant for autoimmunity.
ACKNOWLEDGMENTS
We wish t o acknowledge the excellent technical
contributions of Ms. T. Cummons, the histologic expertise
of Dr. R. Sharma, and the statistical analysis by Ms. H. Su
and Ms. T. Coffey.
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