Soc Nephrol. 9: 1407-1415. 1998. Attenuation of Murine Lupus Nephritis by Mycophenolate. Mofetil. MIEKE C. J. VAN. BRUGGEN,. BIRGITTE. WALGREEN,.
J Am
Attenuation Mofetil
of Murine
MIEKE
C. J. VAN H. M. BERDEN
Jo Division
Abstract.
Mycophenolate
(MMF)
of mycophenolic acid, is effective in prolonging
xenografts.
However,
mechanism
this
study,
the
progression
med.
effect
in the
with
day.
animals of
by
controls
(cumulative
glomerulonephritis
treated
mice
than
and
less
in the
sive
that with
and
clinical
relatively
hydrolyzed
low
by
esterases acid
type
2 isoform
of inosine
sine
monophosphate of de
(MPA).
This
surface
T and B cells, possible
via
(1-4).
since the
MMF
in other
salvage
in signifiin
it has
purine
pathway,
which
of immune
albografts,
but
(reviewed
in references
in inhiof
for
is still
inflicted
by
in Lewis
delayed
occurrence
of acute
rejection
of organ
deterioration
In the effect
of MMF
of action.
December
22, to Dr.
University
Hospital
“St.
1997.
Accepted
Mieke
February
C. J. van
Radboud,”
P.O.
Bruggen, Box
9101,
17,
1998.
Division
both
6500
tis
Journal
Copyright
The
by the
Society American
Society
of Nephmology
to
In
ously
we
to
renal to
perfusion a
decreased
capillary
treat
ongoing
is less
wall
rejection
experience
MMF
was
prevent
the
NZB/W
mice,
function
and
with found
autoimmune
has
in vivo
to
uveoreti-
development
of
MMF
treatment
prolonged
survival
on
the
main
mice
the
mechanism
antibody
formation
of the important
features.
From
an ultimately the
glomerular is instrumental
study,
of
we assessed
MMF
MRL/lpr
terms
on
mouse
disease, of
role
clinical,
immune
(12). binding
basement
For of
for
progresspontane-
which
closely
histologic,
and
the age of 12 to 14 wk, these fatal
gbomerulonephritis the
influence
autoimmune in
glomerulonephritis,
In this
the
on
because
This
SLE
develop
to
in MRL/bpr and
effects
and
(5)
a systemic
human
proliferative
evaluated
progression
MMF
focused
develops
to
study,
glomerubonephritis.
nucleosomes,
of Nephrology
the
in systemic lupus erythematosus (SLE) of tissue lesions, especially gbomerulonephri-
we
of
bodies
l407$03.00/0
of the American tO 1998
Nijmegen,
in MRL/ immunoglob-
beads
diseases.
of renal
and
(10,11),
this
Netherlands. 1046-6673/0908-
HB
Nephrology,
and
used
and
(8).
on disease
in vitro
start of
be
(7)
Because
immunologic Correspondence
albuminuria
1 and 6). There
rats
present
resembles Received
basement
mice compared with suppresses the devel-
(9).
sion
the
antibody
glomerubar
of experimental
rats
in BB
in vivo
effect.
in BALB/c
of glomerubar
autoimmune
diabetes
of cell
specificity not
nitis
primary
in the gbomerular
also
development
of autoantibodies the development
reduces
it can
the
mo-
found
treatment
complexes
not
no immunosup-
albumin. Interestingly, that binding of nucleo-
mice
MMF
CD8
were
nephritis.
MMF. The insights in the mechanism of action of MMF are mainly based on in vitro studies (4,5), and it remains to be determined whether similar mechanisms are responsible for the MMF
that
and
and
and
reduction
and Also,
blood
were
to the
in MMF-treated
binding in lupus
CD4,
hypersensitivity
glomerulonephritis
indicate
inhibit
synthesis is
deposits
to
of the
CD3,
MMF
observed
MMF-treated formation.
Furthermore,
of 90 mg/kg
studies
blood
of glycosylation
groups.
observed
and
results
the
ulin
The
in treating
in reduction
a relative
of bupus
MMF
thereby
has
mice.
opment
is
the action
both
of
complexes
control
and
were
and peripheral
is decreased in MMF-treated It is concluded that MMF
MMF
dehydrogenase.
cells
been
effects
response to methylated bovine serum renal perfusion experiments revealed
orally,
liver,
and inhibition
glycoproteins
was
nodes,
delayed-type
lpr mice.
is immunosuppres-
blocks
and
in
Surprisingly,
inhibition
synthesis
T and B cell proliferation
wall
numbers
properties on
membrane
in MMF-
Furthermore,
and
lymph
some/antinucleosome
wk
immunoglobulin
intestine, MPA
The
0.0001).
of
monophosphate
purine
23 =
0.005).
(MMF)
dehydrogenase
novo
at
P
Administered
in the
mycophenolic
bition
toxicity.
17).
=
a
significantly
transplantation,
mofetil
once
vehicle-treated
0.002).
organ
mycophenolate
(n was
capillary (P
mice
mg/kg)
less severe
amount
glomerular
between
mice
with
=
different pressive
in controls;
(P
the
in MMF-treated
In experimental shown
mice
studies
disease
orally
albuminuria
88%
in spleen,
exam-
h)
histologically
in control
C3 deposits
cantly
of
versus
enlargement
was
alone
pg/l8
spleen
were
1998
Netherlands.
there were no differences between animals with regard to autoantibody
18)
(90
P. M. RIJKE,
immune-modulating
T cells
In
The
no clear-cut
because control
the
TRUUS
Nzjmegen,
of lupus =
compared
was
immunofluorescence
(n
vehicle
incidence
22%
and
diseases.
spontaneous
model
(>300
treatment
mice;
the
mice
received
MMF
MMF-treated
on
WALGREEN,
vivo
9: 1407-1415.
by Mycophenolate
St. Radboud,
morpholin-
the effects
in a vehicle
albuminuria
reduced
about
mouse
dissolved
the
in autoimmune
MMF
MRL/lpr
MMF
Control
incidence
is known
MRL/Ipr
Hospital
which is its active metabsurvival of albografts and
of MMF
of
Eight-week-old
treated
The
little
of action
is
Nephritis
BIRGITTE
University
mofetil
oethyb ester olite. MMF main
BRUGGEN,
Nephrology,
of
Lupus
Soc Nephrol
mice
complex-mediated
the development antinuclear
membrane
(GBM),
of MMF
on proteinuria,
(13-15).
the effect
of
autoanti-
via
1408
Journal
on histologic merubar
lpr
American
severity
addition,
after
both
prophylactic
we
analyzed
the
assessing
the
evaluated
effect
spleen/lymph
node
and
and
main
of
toward
Furthermore,
on
in t’ivo
immune
complexes in predisease
and
serum
and
p=0.0001 80
a)
E 60
C
the
E 40 .0
antibody
(61
(mBSA).
on the binding in a renal
20
of
perfusion
mice. 13
Materials
and
Animals MRL/bpr the University Scripps Clinic Jackson immune
mice
were bred
in the animal
from stock Foundation
(Bar Harbor, ME). by MMF, BALB/c
the Jackson
Laboratory)
and
facilities
originally obtained (La Jolla, CA) For the in vivo mice were used
bred
in our
of
via from
the the
assessment
(also
central
of
obtained
animal
1. Cumulative
mg/kg
body
exceeded
labora-
ter,
a prodrug
Ltd.,
sodium
of MPA,
United
Kingdom)
chloride
polysorbate This
For
the
MMF,
first
set
8-wk-old
mg/kg
body
were
(ii
from
1 7) received
during levels
treatment, blood was drawn toward DNA, nucleosome,
week
The
“surrogate”). nucleosome/Ig by urine
weight)
and
was
considered
300
collection the
for histology.
animals
were
In a second
MMF
on established
ment,
2+).
treated
mild
with
gb(score
J Am
Soc
Nephrol
Figure
3. Glomerular
In addition
tis.
9: 1407-1415,
nuclear
cell
histology.
to this influx
to 3+ scale. some/moderate
This
of the
Deposition incubating
gbomerubonephritis
scale influx;
in
was
microscopy to study
Technika,
the degree
assessed
was
and
performed
deposition
of gbomeruli
of mono-
scored
on a 0
1 + to 2+,
on 2-p.m
of mouse
of mouse
goat
lands)
I :40.
diluted
After
Ig and
cryostat
mouse
(Nordic,
by Ig
diluted
(wt/vol)
BSA.
The
The BALB/c
with
Nether-
United Blinded
investigators. and
The
the mesangium
in at beast
Assessment
25
were
embedded
Kingdom) sections
intensity was
were
per
with
examined
of the
scored
glomerubi
in Aqua-
and examined
by
staining
in the
semiquantitativeby
on
mouse
kidney.
of Immune-Modulating
MRL/lpr
primary antibody (19). In brief, mg/kg
mice
of
adjuvant
ear.
As
left
ear.
mice
S p.1 of
with
17, blood
the anti-mBSA
Vitro
saline
micrometer.
from
on Spleen
day
was given
was
The
of the right
injected
thickness
DTH
48 h after
the
(90
0 until
same
reactivity
is
site
(R)
the rechablenge.
animals
to determine
immunosorbent
Cell
in the
( X I0)
of the antigen-injected
in enzyme-linked
of MMF
of MMF day
the pinna
of ear
site (L) as measured
Effect
previously groups of
in 0. 1 ml of complete
into
measurements
collected
response
dose from
(B).
in normal DTH and
15, a rechablenge
(2 mg/mb)
of the swelling
was
described in two
0. 1 mg of mBSA
an engineer’s
as the ratio
or MMF
also studied of MMF on
a daily
At day
1409
(A)
of vehicle
phosphate-buffered
At 48 h, duplicate
At day
vehicle
received volume
received
and the saline-injected
J
that
subcutaneously
control,
made
with
Mofetil
properties were the influence
subcutaneously.
5 p.1 of mBSA
were
treated
or an equivalent
I 7. At day 4, all mice Freund’s
by Mycophenolate
response according to methods DTH reactivity was determined
BALB/c
orally)
MRUlpr The
Properties
of Lupus
immunosuppressive mice by measuring
lO-wk-obd
C3.
the sections
Tilburg,
the sections
Poole, microscope.
scale
In Vivo
C3c
procedure,
Chemicals,
loops
1%
by incubating
from
expressed
independent
a 0 to 4+
10 mg/mI
containing
studied
anti-mouse
fluorescence
capillary
Belgium)
saline
C3 was
the staining (BDH
a Zeiss
Turnhout,
phosphate-buffered
FITC-babeled
three
score,
loops
was defined as follows: 0, no influx; and 2+ to 3+, extensive influx.
kidneys
Organon
Deposition
mount
photomicrographs
of mouse Ig was studied in direct immunofluorescence the sections with FITC-labebed F(ab), sheep anti-mouse
(Cappeb, I :750
Representative
in capillary
Immunofluorescence sections
Attenuation
1998
assay
Proliferation
(19).
of
Mice in vitro
stimulated
effect
of MMF
mitogenesis
was
of spleen
tested
on concanavalin
cells.
Spleen
cells
A (ConA)from
MRLIIpr
of MMF In the plasma the
samples
Ig concentrations
timatrigel
(anti-GBM)
immunosorbent someflg
of MMF and
the
or vehicle-treated
anti-DNA,
reactivities
assay,
complex
as
assay
were
determined
described was
MRL/bpr
antinucleosome, before
performed
an-
in enzyme-linked (13,14).
as
mice, and
The
described
nucleo-
--
previously
(14,18).
2
The effect number
of
numbers spleen,
of MMF leukocytes
on spleen
in blood,
MRL/lpr (90 mg/kg)
FITC-conjugated thiocyanate-conjugated jugated
rat
anti-mouse
for 30 mm. .
conjugated
rabbit
cells
washed
were
CD8a
hamster .
and
on
T cells in studied in 8-
**
(with
*
tetramethybrhodamine isoCD3 or with FITC-con-
CD3
was
Ig (DAKO, times
nodes,
tetramethybrhodamine
for B cells
.
three
CD4 and anti-mouse and
anti-mouse
lymph
on
for 4 wk (,z = 6) and 7 wk (n = 3) Cells (5 X l0) were incubated with
anti-mouse
Staining
and
node weight,
CD8, and CD4/CD8 peripheral blood was
mice treated or vehicle.
rat anti-mouse hamster
nate-conjugated
and lymph
spleen,
of B cells, CD3, CD4, axilbary lymph nodes, and
to 9-wk-old with MMF
CA)
treatment
isothiocya-
o
(Pharmingen,
San
Diego,
performed
using
FITC-
Denmark).
The
Gbostrup,
phosphate-buffered
saline,
Ig
glomerular 4. The amount loops in gbomerubi of significant decrease in in MMF-treated mice
I%
Figure
BSA, pH 7.4) and analyzed using a Coulter Epics XL flow cytometer (Coubter, Hialeah, FL). The percentage of CD4/CD8 T cells was calculated as follows: %(CD4/CD8) = %(CD3 ) [%(CD3/ CD4) + %(CD3/CD8)].
0.0017;
-
C3
**
0.0002.
deposits
.
of Ig and C3 deposits in glomerular capillary mice treated with MMF () or vehicle (0). A the amount of Ig and C3 deposits was observed compared with vehicle-treated controls. *P
1410
Journal
5. Representative
Figure
treatment
mice
(n
3; age,
=
l0
cells/well
7 to 8 wk)
findings
incubated
(Sigma)
concentration
MRL/lpr
for 72 h at 37#{176}C in a 5%
in a dose
(mg/mb) and
mice
mycophenobate
of I .25 p.g/mb.
and
and
mofetil
and Treatment (wk)
Prolifer-
after
(MMF)
titers
in plasma
treatment
with
of
MMF
3.7
±
1.2
3.2
±
1.0
4.2
±
1.3
4.1
±
1.1
6
6.6±2.6
5.9±2.1
9
8.9
8.9
20.0
4.7
± 5.1
19.1
0
1365
±
3
4471
± 651
6 9
7059 10,773
±
1358
±
12
12,141
± 4.7 ± 6.1
109
1555
±
147
5155
±
1346
6267
± 887
2198
8355
±
1517
± 2212
15,555
±
2212
in
Control
0
644±50 2400 ± 368
788±136
3
2071
± 254
6 9
5918 6035
±
577
4222
± 701
±
1115
7688
±
12
7764
±
1909
13,244
2680
± 3264
Antimatrigel
0
72±8
92±8
3
264±34
187±22
6 9
321±31 400±50
276±40 316±45
12
413±83
390±54
for
any
±
SD. There
parameter
were
at any
no statistically
time
assessed
deposition
1407-1415,
1998
of Ig after
vehicle
by incorporation during
of tritiated
the last
MRL/lpr a vehicle
mice (90
mice
renal
(n
4) were
=
mg/kg
(n
study,
treated
wt),
4) received
=
perfusion
body
once
equal
the
for
Blinded The
point.
antibody
mAb
34 were
was performed as described kidney was taken out and
nitrogen
evaluation
sections
amount
mesangium
(1
of
were
and
intensity
were
scored
with
MMF
a day,
for
5 wk.
were
of vehicle
anesthetized
by
(Narcovet, Apantibodies of the
obtained
by purification
of
as described antinucleosome
(13). an-
before (13). After perfusion, the immediately snap-frozen in liquid
IgG2a
examined
orally amounts
mice
supematant under physiologic conditions, perfusion of these nucleosome-complexed
tibodies perfused
thymidine
I 8 h of incubation.
of Nucleosome-Complexed Antibody
MRL/lpr For
binding
by
by three
immunofluorescence.
independent
investigators.
of the staining
in the capillary
semiquantitativeby
on
loops
a 0 to 3+
and the scale,
as
follows: 0, no staining; 1 +, mild staining; 2+, moderate 3+, strong staining. For the staining of GBM-heparan
staining; and sulfate (HS),
indirect
to techniques
immunofluorescence
described
previously
Statistical
Antinucleosome
as mean
showing
concentration)
antinucleosomal
3
are given
mice
intraperitoneal administration of sodium pentobarbitab pharma, Arnhem, The Netherlands). Complexed
concentration
differences
dissolved
culture Renal
±
was
final
alone.
or vehiclea
Vehicle
in wells
p.Ci/mb,
Six-week-old
0
Results
of MRLIbpr
Renal Perfusion Antinucleosome
Anti-DNA
a
ation
anti-DNA,
antimatrigel
before
12
significant
of glomerubi
9:
(B).
were
antinucleosome,
IgG
J Am Soc Nephrol
of Nephrology
of the immunofluorescence
treatment
by ConA
1. IgG
Parameter
Society
in 96-well microtiter plates at a concentration of 5 X in the presence or absence of MMF (0 to S p.M). Cells
stimulated
Table
American
examples
(A) or MMF
CO, atmosphere were
of the
was
performed,
according
(17).
Analyses
Statistical
analyses
were
performed
using
the
Mann-Whitney
U
test.
Results Effects
of MMF
on
Mice: Attenuation of Nephritis In the significantly vehicle-treated
preventive
the
Disease
was
whereas
the
the administration
and
dose
of 90
of abbuminuria
0.0001)
of albuminuria in
in MRU1pr
Symptoms
a daily onset
(P
controls
incidence
Furthermore,
study,
reduced
cumulative 88%,
Glomerular of Clinical
(Figure
Severity mg/kg
MMF
compared
with
1). At 23 wk,
in vehicle-treated
MMF-treated
of MMF
group
significantly
the
animals it
was
22%.
reduced
J Am
Soc Nephrol
Table
2.
9: 1407-1415,
Effects
after
Attenuation
1998
4 wk (n
6) and
7 wk
3) of treatment
(n
of MRLIlpr
of Lupus
mice
by Mycophenolate
with
MMF
Mofetil
or vehicbea
Wee k 4
Wee k 7
Parameter Vehicle Weight
MMF
Vehicle
MMF
(mg)
spleen
170 ± 20
lymph node
57
190 ± 31
3
±
57 ±
265
10
± 99
69 ±
255
10
50
±
65 ± 9
Leukocytes spleen
X
lymph
node
l0
blood
X
13.1 X
i0
106/mb
15.0
± 3.2
12.3
±
10
17.0
± 7.1
3.3
± 4.6 ±
1.0
4.4
± 3.8
4.1
±
1.9
4.2
± 3.1
2.5
±
1.5
2.1
±
2.2
±
1.7
1.7
±
36.1
±
10
41.0±11
30.3
± 9.1
1.5
1.4
% B cells spleen
41.2±
lymph
node
26.5
blood
lymph
± 4.8
5.5
10.8
± 8.7
13.3
1.8 ± 2.1
lymph
node
blood % CD8
node
blood CD4/CD8
significant
differences
MMF-
and
(Figures control
2 and MRL/bpr
of the
18 mice
were
14.3
±
6.2
10
29.0
±
Il
±
2.1
4.0
± 2.3
1.8 ± 2.1
4.0
20.8
± 6.4
19.0
± 9.1
17.0
± 6.9
34.3
± 5.1
31.2
± 9.9
27.7
± 10
29.7
± 4.1
32.3
± 2.9
25.3
± 6.8
30.0
± 3.5
± 4.2
7.8
± 6.1
4.1
20.2
±
20.9
± 3.3
9.8
± 2.1
9.3
± 1.4
8.8
23.0
± 5.7
23.8
± 5.6
18.1
±
17.7
± 4.7
21.8
± 2.9
16.8
± 5.0
2.2
± 0.7
2.3
± 0.6
2.1
± 0.9
mice,
treated
MMF
with
the
for
1 .4 ± 0.4
1.7 ± 0.4
1.5
1.5 ± 0.6
1.4 ± 0.3
2.4
0.005)
=
(P